Elongase gene and production of Δ9-polyunsaturated fatty acids

ABSTRACT

This invention relates to a new elongase gene having the sequence SEQ ID NO: 1 or its derivatives, to a gene construct comprising this sequence or its derivatives and to its use. The inventive nucleic acid sequence encodes a polypeptide which elongates α-linolenic acid (C 18:3 d9, 12, 15 ) by at least two carbon atoms whereas γ-linolenic acid (C 18:3 d6, 9, 12 ) is not elongated. The invention additionally relates to vectors or organisms comprising an elongase gene having the sequence SEQ ID NO: 1 or its derivatives. 
     The invention further relates to a process for the production of polyunsaturated fatty acids (=PUFAs) with an organism which comprises the elongase gene and which organism produces high amounts of oils and especially oils with a high content of unsaturated fatty acids. Additionally the invention relates to an oil and/or fatty acid composition with a higher content of polyunsaturated C 20  or C 22  fatty acids with at least two double bonds and/or to a triacylglycerol composition with a higher content of said polyunsaturated fatty acids.

FIELD OF THE INVENTION

This invention relates to a new elongase gene having the sequence SEQ IDNO: 1 or its derivatives, to a gene construct comprising this sequenceor its derivatives and to its use. The inventive nucleic acid sequenceencodes a polypeptide which elongates α-linolenic acid(C_(18:3 d9, 12, 15)) by at least two carbon atoms whereas γ-linolenicacid (C_(18:3 d6, 9, 12)) is not elongated. The invention additionallyrelates to vectors or organisms comprising an elongase gene having thesequence SEQ ID NO: 1 or its derivatives.

The invention further relates to a process for the production ofpolyunsaturated fatty acids (=PUFAs) with an organism which comprisesthe elongase gene and which organism produces high amounts of oils andespecially oils with a high content of unsaturated fatty acids.Additionally the invention relates to an oil and/or fatty acidcomposition with a higher content of polyunsaturated C₂₀- or C₂₂-fattyacids with at least two double bonds and/or to a triacylglycerolcomposition with a higher content of said polyunsaturated fatty acids.

BACKGROUND OF THE INVENTION

Certain products and by-products of naturally-occurring metabolicprocesses in cells have utility in a wide array of industries, includingthe food, feed, cosmetics, and pharmaceutical industries. Thesemolecules, collectively termed ‘fine chemicals’, also include lipids andfatty acids whereof one representative class of molecules ispolyunsaturated fatty acids. Polyunsaturated fatty acids (=PUFAs) areadded for example to infant formulas to create a higher nutrition valueof such formulas. PUFAs have for example a positive influence on thecholesterol level of the blood in humans and therefore are useful in theprotection against heart diseases. Fine chemicals and polyunsaturatedfatty acids (=PUFAs) can be isolated from animal sources such as forexample fish or produced with microorganisms through the large-scalefermentation of microorganisms developed to produce and accumulate orsecrete large quantities of one or more desired molecules.

Particularly useful microorganisms for the production of PUFAs aremicroorganisms such as the algae Isochrysis galbana, Phaedactylumtricornutum or Crypthecodinium species, ciliates like Stylonychia orColpidium, fungi like Mortierella, Entomophthora, Mucor orThrausto-/Schizochytrium species. Through strain selection, a number ofmutant strains of the respective microorganisms have been developedwhich produce an array of desirable compounds including PUFAs. However,selection of strains improved for the production of a particularmolecule is a time-consuming and difficult process.

Alternatively the production of fine chemicals can be most convenientlyperformed via the large scale production of plants developed to produceaforementioned PUFAs. Particularly well suited plants for this purposeare oilseed plants containing high amounts of lipid compounds such asrapeseed, canola, linseed, soybean, sunflower, borage and eveningprimrose. But also other crop plants containing oils or lipids and fattyacids are well suited as mentioned in the detailed description of thisinvention. Through conventional breeding, a number of mutant plants havebeen developed which produce an array of desirable lipids and fattyacids, cofactors and enzymes. However, selection of new plant cultivarsbred for the production of a particular molecule is a time-consuming anddifficult process or even impossible if the compound does not naturallyoccur in the respective oil crop as in the case of C₂₀ and higher carbonchain polyunsaturated fatty acids.

SUMMARY OF THE INVENTION

This invention provides a novel nucleic acid molecule as described inSEQ ID NO: 1 which may be used to modify oils, fatty acids, lipids,lipid derived compounds and most preferred to produce polyunsaturatedfatty acids.

Microorganisms such as Mortierella, Entomophthora, Mucor,Crypthecodinium as well as other algae and fungi and plants, especiallyoilseed plants, are commonly used in industry for the large-scaleproduction of a variety of fine chemicals.

Given the availability of cloning vectors and techniques for geneticmanipulation of the abovementioned microorganisms and ciliates such asdisclosed in WO 98/01572 or algae and related organisms such asPhaeodactylum tricornutum described in Falciatore et al., 1999, MarineBiotechnology 1 (3):239-251 as well as Dunahay et al. 1995, Genetictransformation of diatoms, J. Phycol. 31:10004-1012 and referencestherein, the nucleic acid molecules of the invention may be utilized inthe genetic engineering of these organisms to make them better or moreefficient producers of one or more fine chemicals. This improvedproduction or efficiency of production of a fine chemical may be due toa direct effect of manipulation of a gene of the invention, or it may bedue to an indirect effect of such manipulation.

Mosses and algae are the only known plant systems that produceconsiderable amounts of polyunsaturated fatty acids like arachidonicacid (ARA) and/or eicosapentaenoic acid (EPA) and/or docosahexaenoicacid (DHA). Therefore nucleic acid molecules originating from an algalike Isochrysis galbana are especially suited to modify the lipid andPUFA production system in a host, especially in microorganisms such asthe abovementioned microorganisms and plants such as oilseed plants, forexample rapeseed, canola, linseed, soybean, sunflower, borage.Furthermore nucleic acids from the alga Isochrysis galbana can be usedto identify those DNA sequences and enzymes in other species which areuseful to modify the biosynthesis of precursor molecules of PUFAs in therespective organisms.

The alga Isochrysis galbana shares a high degree of homology on the DNAsequence and polypeptide levels with other algae allowing the use ofheterologous screening of DNA molecules with probes evolving from otheralgae or organisms, thus enabling the derivation of a consensus sequencesuitable for heterologous screening or functional annotation andprediction of gene functions in third species. The ability to identifysuch functions can therefore have significant relevance, e.g.,prediction of substrate specificity of enzymes. Further, these nucleicacid molecules may serve as reference sequences for the mapping of otheralgae or for the derivation of PCR primers.

These novel nucleic acid molecules can encode proteins referred toherein as PUFA specific elongases (PSEs or singular PSE). These PSEs arecapable of, for example, performing a function involved in themetabolism (e.g., the biosynthesis or degradation) of compoundsnecessary for lipid or fatty acid biosynthesis like PUFAs, or ofassisting in the transmembrane transport of one or more lipid/fatty acidcompounds either into or out of the cell.

In the present application we show the function of one of thesesequences in more detail. We have isolated for the first time afunctionally active gene encoding a highly specific elongase activitysuitable to produce long chain unsaturated fatty acids from α-linolenicacid (C_(18:3 d9, 12, 15)) while γ-linolenic acid (C_(18:3 d6, 9, 12))is not elongated. We will herein therefore refer to an ASE(“alpha-linolenic acid specific elongase”) gene or protein thusrepresenting an enzymatic activity leading to the elongation of omega-3fatty acids or delta-9 desaturated long chain polyunsaturated fattyacids at least by two carbon atoms. Other publications and patents havenot been able before to show a functionally active ASE gene that isspecific for α-linolenic acid (ALA) and which does not acceptγ-linolenic acid (GLA) as a substrate though there are various patentapplications known showing the elongation of short or medium chainsaturated fatty acids (WO 98/46776 and U.S. Pat. No. 5,475,099). WO00/12720 describes various PSEs from various organisms but none of thedescribed genes was shown to be specific for ALA while discriminatingagainst GLA. Genes shown to encode PSEs from WO 00/12720 all accept GLAas a substrate, hence these enzymes lead to the elongation of Δ6desaturated long chain polyunsaturated fatty acids but not of Δ9desaturated fatty acids as disclosed in the present invention.

The unique feature of the ASE disclosed in the present invention isimportant as resulting products are limited to desired products whilebeing free of unwanted PUFA molecules such as those resulting from theelongation of GLA.

WO 99/64616, WO 98/46763, WO 98/46764, WO 98/46765 describe theproduction of PUFAs in transgenic plants showing the cloning andfunctional expression of respective Δ12-, Δ5- or Δ6-desaturaseactivities from several sources lacking the demonstration of an ASEencoding gene or an α-linolenic acid specific Δ6-desaturase gene andfunctional activity necessary for the production of eicosapentaenoicacid and related precursors from ALA.

For the production of PUFAs it is necessary that the polyunsaturatedfatty acid molecules such as the preferred C₁₈ fatty acids are elongatedby at least two carbon atoms by the enzymatic activity of an elongase.The nucleic acid sequence of the invention encodes the first elongasederived from a plant which has the ability to elongateC_(18:3 d6, 9, 12) fatty acids with at least two double bonds,preferably three double bonds, in the fatty acid by at least two carbonatoms. After one round of elongation this enzymatic activity leads toC₂₀ fatty acids and after two, three and four rounds of elongation toC₂₂, C₂₄ or C₂₆ fatty acids. With the elongase of the invention it isalso possible to synthesize longer PUFAs. The activity of the elongaseof the invention leads preferably to C₂₀ and/or C₂₂ fatty acids with atleast two double bonds in the fatty acid molecule, preferably with threeor four double bonds, particularly preferably three double bonds, in thefatty acid molecule. Preferred fatty acid molecules of the elongationare fatty acid molecules with a double bond in Δ9-postion. After theelongation with the inventive enzyme has taken place furtherdesaturation steps might occur. Therefore the products of the elongaseactivity and the possible further desaturation lead to preferred PUFAswith a higher desaturation grade such as docosadienoic acid, arachidonicacid, ω6-eicosatrienoic acid, dihomo-γ-linolenic acid, eicosapentenoicacid, ω3-eicosatrienoic acid, ω3-eicosatetraenoic acid, docosapentaenoicacid or docosahexaenoic acid. A particularly preferred fatty acid is theelongation product stearidonic acid of the α-linolenic acid(C_(18:3 d9, 12, 15)). Substrates of the enzymatic activity of theinvention are preferably Δ9 desaturated fatty acids which have the firstdouble bond in Δ9-postion such as axillarenic acid, vernolic acid (C₁₈,Δ9_(cis), 12-13_(epoxy)), conjugated linoleic acid (C₁₈, Δ9_(cis),11_(trans)), sterolic acid (C₁₈, Δ9-acetylenic), α-parinaric acid (C₁₈,Δ9_(cis), 11_(trans), 13_(trans)), palmitoleic acid (C₁₈, Δ9cis),linoleic acid or α-linolenic acid. Preferred substrates are linoleicacid and/or α-linolenic acid. The fatty acids with at least two doublebonds in the fatty acid can be elongated by the enzymatic activity ofthe invention in the form of the free fatty acids, the acyl-CoA-fattyacids, alkyl esters of the fatty acids or in the form of the esters suchas phospholipids, glycolipids, sphingolipids, phosphoglycerides,monoacylglycerides, diacylglycerides or triacylglycerides, preferably inthe form of free fatty acid or the acyl-CoA-fatty acids.

Given the availability of cloning vectors for use in plants and planttransformation, such as those published in and cited therein: PlantMolecular Biology and Biotechnology (CRC Press, Boca Raton, Fla.),chapter 6/7, p. 71-119 (1993); F. F. White, Vectors for Gene Transfer inHigher Plants; in: Transgenic Plants, Vol. 1, Engineering andUtilization, eds.: Kung and R. Wu, Academic Press, 1993, 15-38; B. Jeneset al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1,Engineering and Utilization, eds.: Kung and R. Wu, Academic Press(1993), 128-143; Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol.42 (1991), 205-225)), the nucleic acid molecules of the invention may beutilized in the genetic engineering of a wide variety of plants to makethem better or more efficient producers of one or more lipid derivedproducts such as PUFAs. This improved production or efficiency ofproduction of a lipid derived product such as PUFAs may be due to adirect effect of manipulation of a gene of the invention, or it may bedue to an indirect effect of such manipulation.

There are a number of mechanisms by which the alteration of an ASEprotein of the invention may directly affect the yield, production,and/or efficiency of production of a fine chemical from an oilseed plantor microorganism due to such an altered protein. The ASE protein or genemay be increased in number or activity such that greater quantities ofthese compounds are produced or the compound may be produced de novo asthe organisms were lacking this activity and capacity of biosynthesisbefore the introduction of the respective gene.

The introduction of an ASE gene into an organism or cell may not justincrease the biosynthetic flux into an end product, it may also increaseor create de novo the respective triacylglycerol composition. Similarly,other genes involved in the import of nutrients necessary for thebiosynthesis of one or more fine chemicals (e.g., fatty acids, polar andneutral lipids) may be increased in number or activity such that theseprecursors, cofactors, or intermediate compounds are increased inconcentration within the cell or within the storing compartment, thusincreasing further the capability of the cell to produce PUFAs asdescribed below. Fatty acids and lipids themselves are desirable finechemicals; by optimizing the activity or increasing the number of one ormore ASEs which participate in the biosynthesis of these compounds, orby impairing the activity of one or more ASEs which are involved in thedegradation of these compounds, it may be possible to increase theyield, production, and/or efficiency of production of fatty acid andlipid molecules from plants or microorganisms.

The mutagenesis of the ASE gene of the invention may also result in anASE protein having altered activities which directly or indirectlyimpact the production of one or more desired fine chemicals. For examplethe ASE gene of the invention may be increased in number or activitysuch that the normal metabolic wastes or byproducts of the cell(possibly increased in quantity due to the overproduction of the desiredfine chemical) are efficiently exported before they are able to destroyother molecules or processes within the cell (which would decrease theviability of the cell) or to interfere with fine chemical biosyntheticpathways (which would decrease the yield, production, or efficiency ofproduction of the desired fine chemical). Further, the relatively largeintracellular quantities of the desired fine chemical may in themselvesbe toxic to the cell or may interfere with enzyme feedback mechanismssuch as allosteric regulation; for example, by increasing the activityor number of other downstream enzymes or detoxifying enzymes of the PUFApathway, it might increase the allocation of the PUFA into thetriacylglycerol fraction, one might increase the viability of seedcells, in turn leading to a better development of cells in the cultureor in a seed producing the desired fine chemical. The ASE gene of theinvention may also be manipulated such that the relative amounts ofdifferent lipid and fatty acid molecules are produced. This may have aprofound effect on the lipid composition of the membrane of the cell andwould create novel oils in addition to the occurrence of newlysynthesized PUFAs. Since each type of lipid has different physicalproperties, an alteration in the lipid composition of a membrane maysignificantly alter membrane fluidity. Changes in membrane fluidity canimpact the transport of molecules across the membrane, as well as theintegrity of the cell, both of which have a profound effect on theproduction of fine chemicals. In plants these changes can moreover alsoinfluence other characteristics like tolerance towards abiotic andbiotic stress conditions.

Biotic and abiotic stress tolerance is a general trait desired to beinherited into a wide variety of plants like maize, wheat, rye, oats,triticale, rice, barley, soybean, peanut, cotton, rapeseed and canola,manihot, pepper, sunflower and tagetes, solanaceous plants like potato,tobacco, eggplant, and tomato, Vicia species, pea, alfalfa, bushy plants(coffee, cacao, tea), Salix species, trees (oil palm, coconut) andperennial grasses and forage crops. These crop plants are also preferredtarget plants for genetic engineering as one further embodiment of thepresent invention. Particularly preferred plants of the invention areoilseed plants such as soybean, peanut, rapeseed, canola, sunflower,safflower, trees (oil palm, coconut) or crops such as maize, wheat, rye,oats, triticale, rice, barley, alfalfa or bushy plants (coffee, cacao,tea).

In another embodiment, the isolated nucleic acid molecule encodes aprotein or portion thereof wherein the protein or portion thereofincludes an amino acid sequence which is sufficiently homologous to anamino acid sequence of sequence SEQ ID NO: 2 such that the protein orportion thereof maintains an ASE activity. Preferably, the protein orportion thereof encoded by the nucleic acid molecule maintains theability to participate in the metabolism of compounds necessary for theconstruction of PUFAs or cellular membranes of plants or in thetransport of molecules across these membranes. In one embodiment, theprotein encoded by the nucleic acid molecule is at least about 50%,preferably at least about 60%, and more preferably at least about 70%,80%, or 90% and most preferably at least about 95%, 96%, 97%, 98%, or99% or more homologous to an amino acid sequence of sequence SEQ ID NO:2. In another preferred embodiment, the protein is a full lengthIsochrysis galbana protein which is substantially homologous to anentire amino acid sequence of SEQ ID NO: 2 (derived from an open readingframe shown in SEQ ID NO: 1).

Accordingly, one aspect of the invention pertains to isolated nucleicacid molecules (e.g., cDNAs) comprising a nucleotide sequence encodingan ASE protein or biologically active portions thereof, as well asnucleic acid fragments suitable as primers or hybridization probes forthe detection or amplification of ASE-encoding nucleic acids (e.g., DNAor mRNA). In particularly preferred embodiments, the nucleic acidmolecule comprises one of the nucleotide sequences set forth in SEQ IDNO: 1, derivatives of said sequence or the coding region or a complementor enzymatically active part thereof. In other particularly preferredembodiments, the isolated nucleic acid molecule of the inventioncomprises a nucleotide sequence which hybridizes to or is at least about50%, preferably at least about 60%, more preferably at least about 70%,80% or 90%, and even more preferably at least about 95%, 96%, 97%, 98%,99% or more homologous to a nucleotide sequence as in SEQ ID NO: 1,derivatives or a portion thereof. In other preferred embodiments, theisolated nucleic acid molecule encodes an amino acid sequence as setforth in SEQ ID NO: 2. The preferred ASE gene of the present inventionalso preferably possesses at least one of the ASE activities describedherein.

In another preferred embodiment, the isolated nucleic acid molecule isderived from Isochrysis galbana and encodes a protein (e.g., an ASEfusion protein) which includes a biologically active domain which is atleast about 50% or more homologous to one of the amino acid sequences ofSEQ ID NO: 2 and is able to participate in the metabolism of compoundsnecessary for the construction of cellular membranes or in the transportof molecules across these membranes, or has one or more of theactivities set forth in Tab. 2, and which also includes heterologousnucleic acid sequences encoding a heterologous polypeptide or regulatoryregions.

In another embodiment, the isolated nucleic acid molecule is at least 15nucleotides in length and hybridizes under stringent conditions with anucleic acid molecule comprising a nucleotide sequence of SEQ ID NO: 1.Preferably, the isolated nucleic acid molecule corresponds to anaturally-occurring nucleic acid molecule. More preferably, the isolatednucleic acid encodes a naturally-occurring Isochrysis galbana ASE, or abiologically active portion thereof.

Another aspect of the invention pertains to vectors, e.g., recombinantexpression vectors, containing the nucleic acid molecules of theinvention, and host cells into which such vectors have been introduced,especially microorganisms, plant cells, plant tissue, plant organs orwhole plants. In one embodiment, such a host cell is a cell capable ofstoring fine chemical compounds, especially PUFAs, in order to isolatethe desired compound from harvested material. The compound (oils,lipids, triacyl glycerides, fatty acids) or the ASE can then be isolatedfrom the medium or the host cell, which in plants are cells containingand storing fine chemical compounds, most preferably cells of storagetissues like seed coats, tubers, epidermal and seed cells.

Yet another aspect of the invention pertains to an isolated ASE geneshown in SEQ ID NO: 1 or a portion thereof, e.g., a biologically activeportion thereof. In a preferred embodiment, the isolated ASE or portionthereof can participate in the metabolism of compounds necessary for theconstruction of cellular membranes in a microorganism or a plant cell,or in the transport of molecules across its membranes. In anotherpreferred embodiment, the isolated ASE or portion thereof issufficiently homologous to an amino acid sequence of SEQ ID NO: 2 suchthat the protein or portion thereof maintains the ability to participatein the metabolism of compounds necessary for the construction ofcellular membranes in microorganisms or plant cells, or in the transportof molecules across these membranes.

Hence in another preferred embodiment, the alga Isochrysis galbana canbe used to show the function of a moss gene using homologousrecombination based on the nucleic acids-described in this invention.

Still another aspect of the invention pertains to an isolated ASE geneor a portion, e.g., a biologically active portion, thereof. In apreferred embodiment, the isolated ASE or portion thereof canparticipate in the metabolism of compounds necessary for theconstruction of cellular membranes in a microorganism or a plant cell,or in the transport of molecules across its membranes. In anotherpreferred embodiment, the isolated PSE or portion thereof issufficiently homologous to an amino acid sequence of SEQ ID NO: 2 suchthat the protein or portion thereof maintains the ability to participatein the metabolism of compounds necessary for the construction ofcellular membranes in microorganisms or plant cells, or in the transportof molecules across these membranes.

The invention also provides an isolated preparation of an ASE. Inpreferred embodiments, the ASE gene comprises an amino acid sequence ofSEQ ID NO: 2. In another preferred embodiment, the invention pertains toan isolated full length protein which is substantially homologous to anentire amino acid sequence of SEQ ID NO: 2 (encoded by an open readingframe set forth in SEQ ID NO: 1). In another embodiment, the protein isat least about 50%, preferably at least about 60%, and more preferablyat least about 70%, 80%, or 90%, and most preferably at least about 95%,96%, 97%, 98%, or 99% or more homologous to an entire amino acidsequence of SEQ ID NO: 2. In other embodiments, the isolated ASEcomprises an amino acid sequence which is at least about 50% or morehomologous to one of the amino acid sequences of SEQ ID NO: 2 and isable to participate in the metabolism of compounds necessary for theconstruction of fatty acids in a microorganism or a plant cell, or inthe transport of molecules across these membranes, or has one or more ofthe PUFA elongating activities, thus meaning the elongation of C18carbon chains being desaturated bearing at least two double bondpositions.

Alternatively, the isolated ASE can comprise an amino acid sequencewhich is encoded by a nucleotide sequence which hybridizes, e.g.,hybridizes under stringent conditions, or is at least about 50%,preferably at least about 60%, more preferably at least about 70%, 80%,or 90%, and even more preferably at least about 95%, 96%, 97%, 98%, or99% or more homologous, to a nucleotide sequence of SEQUENCE ID NO: 1.It is also preferred that the preferred forms of ASEs also have one ormore of the ASE activities described herein.

The ASE polypeptide, or a biologically active portion thereof, can beoperatively linked to a non-ASE polypeptide to form a fusion protein. Inpreferred embodiments, this fusion protein has an activity which differsfrom that of the ASE alone. In other preferred embodiments, this fusionprotein participates in the metabolism of compounds necessary for thesynthesis of lipids and fatty acids, cofactors and enzymes inmicroorganisms or plants, or in the transport of molecules across themembranes of plants. In particularly preferred embodiments, integrationof this fusion protein into a host cell modulates production of adesired compound from the cell. In a preferred embodiment such fusionproteins also contain Δ4-, Δ5- or Δ8-desaturase activities alone or incombination. Especially a Δ8-desaturase from Euglena gracilis describedin WO 00/34439 and a Δ5-desaturase gene described in U.S. Pat. No.6,051,754 (M. alpina), GB9814034.6 (C. elegans) or a Δ12- andΔ15-desaturase gene described in U.S. Pat. No. 5,850,026 are suitablegenes for coexpression with an ASE gene of the present invention. Noneof the cited patents shows coexpression with an ASE gene described inthe present invention.

Another aspect of the invention pertains to a method for producing afine chemical. This method involves either the culturing of a suitablemicroorganism or culturing plant cells, tissues, organs or whole plantscontaining a vector directing the expression of an ASE nucleic acidmolecule of the invention, such that a fine chemical is produced. In apreferred embodiment, this method further includes the step of obtaininga cell containing such a vector, in which a cell is transformed with avector directing the expression of an ASE nucleic acid. In anotherpreferred embodiment, this method includes the step of recovering thefine chemical from the culture. In a particularly preferred embodiment,the cell is from an alga such as Phaeodactylum, ciliates such asColpidium or Stylonichia, fungi such as Mortierella or Thraustochytriumor Schizochytrium or from oilseed plants as mentioned above.

Another aspect of the invention pertains to methods for modulatingproduction of a molecule from a microorganism. Such methods includecontacting the cell with an agent which modulates ASE activity or ASEnucleic acid expression such that a cell associated activity is alteredrelative to this same activity in the absence of the agent. In apreferred embodiment, the cell is modulated for one or more metabolicpathways for lipids and fatty acids, cofactors and enzymes or ismodulated for the transport of compounds across such membranes, suchthat the yields or rates of production of a desired fine chemical bythis microorganism are improved. The agent which modulates ASE activitycan be an agent which stimulates ASE activity or ASE nucleic acidexpression. Examples of agents which stimulate ASE activity or ASEnucleic acid expression include small molecules, active ASEs, andnucleic acids encoding ASEs that have been introduced into the cell.Examples of agents which inhibit ASE activity or expression includesmall molecules and antisense ASE nucleic acid molecules.

Another aspect of the invention pertains to methods for modulatingyields of a desired compound from a cell, involving the introduction ofa wild-type or mutant ASE gene into a cell, the gene being eithermaintained on a separate plasmid or integrated into the genome of thehost cell. If integrated into the genome, such integration can berandom, or it can take place by recombination such that the native geneis replaced by the introduced copy, causing the production of thedesired compound from the cell to be modulated or by using a gene intrans such that the gene is functionally linked to a functionalexpression unit containing at least a sequence facilitating theexpression of a gene and a sequence facilitating the polyadenylation ofa functionally transcribed gene.

In a preferred embodiment, said yields are modified. In anotherpreferred embodiment, said desired chemical is increased while unwanteddisturbing compounds can be decreased. In a particularlypreferred-embodiment, said desired fine chemical is a lipid or fattyacid, cofactor or enzyme. In an especially preferred embodiment, saidchemical is a polyunsaturated fatty acid. More preferably it is chosenfrom arachidonic acid (ARA), eicosapentaenoic acid (EPA) ordocosahexaenoic acid (DHA).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: shows the pairwise alignment of Isochrysis galbana elongase(Ig_ASE1, upper row) with Mortierella alpina elongase (SEQ ID NO:14)(M.AlpinaGlelo. lower row). Identities are shown in bold characters.

FIG. 2: shows the pairwise alignment of polypeptides of Ig_ASE1 (upperrow) and mouse (SEQ ID NO:15) (lower row). Identities are shown in boldcharacters.

FIGS. 3A-3D illustrate HPLC chromatograms of fatty acid methyl estersisolated from yeast expressing the Ig_ASE1 gene product.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides ASE nucleic acid and protein moleculeswhich are involved in the metabolism of lipids and fatty acids, PUFAcofactors and enzymes in the alga Isochrysis galbana or in the transportof lipophilic compounds across membranes. The molecules of the inventionmay be utilized in the modulation of production of fine chemicals frommicroorganisms, such as ciliates such as Colpidium or Stylonichia, fungisuch as Mortierella or Thraustochytrium or Schizochytrium, algae such asPhaeodactylum, and/or plants like maize, wheat, rye, oats, triticale,rice, barley, soybean, peanut, cotton, Brassica species like rapeseed,canola and turnip rape, linseed, pepper, sunflower, borage, eveningprimrose and tagetes, solanaceous plants like potato, tobacco, eggplant,and tomato, Vicia species, pea, manihot, alfalfa, bushy plants (coffee,cacao, tea), Salix species, trees (oil palm, coconut) and perennialgrasses and forage crops either directly (e.g., where overexpression oroptimization of a fatty acid biosynthesis protein has a direct impact onthe yield, production, and/or efficiency of production of the fatty acidfrom modified organisms), or may have an indirect impact whichnonetheless results in an increase of yield, production, and/orefficiency of production of a desired compound or decrease of undesiredcompounds (e.g., where modulation of the metabolism of lipids and fattyacids, cofactors and enzymes results in alterations in the yield,production, and/or efficiency of production or the composition ofdesired compounds within the cells, which in turn may impact theproduction of one or more fine chemicals). Aspects of the invention arefurther explicated below.

I. Fine Chemicals and PUFAs

The term ‘fine chemical’ is art-recognized and includes moleculesproduced by an organism which have applications in various industries,such as, but not limited to, the pharmaceutical, agriculture, food, feedand cosmetics industries. Such compounds also include lipids, fattyacids, cofactors and enzymes etc. (as described e.g. in Kuninaka, A.(1996) Nucleotides and related compounds, p. 561-612, in Biotechnologyvol. 6, Rehm et al., eds. VCH: Weinheim, and references containedtherein), lipids, both saturated and polyunsaturated fatty acids (e.g.,arachidonic acid), vitamins and cofactors (as described in Ullmann'sEncyclopedia of Industrial Chemistry, vol. A27, Vitamins, p. 443-613(1996) VCH Weinheim and references therein; and Ong, A. S., Niki, E. &Packer, L. (1995) Nutrition, Lipids, Health, and Disease Proceedings ofthe UNESCO/Confederation of Scientific and Technological Associations inMalaysia, and the Society for Free Radical Research, Asia, held Sep.1-3, 1994 at Penang, Malaysia, AOCS Press, (1995)), enzymes, and allother chemicals described by Gutcho (1983) in Chemicals by Fermentation,Noyes Data Corporation, ISBN: 0818805086 and references therein. Themetabolism and uses of certain of these fine chemicals are furtherexplicated below.

The combination of various precursor molecules and biosynthetic enzymesresults in the production of different fatty acid molecules, which has aprofound effect on the composition of the membrane. It can be assumedthat PUFAs will not just be incorporated into triacylglycerol but alsointo membrane lipids.

The synthesis of membranes is a well-characterized process involving anumber of components including lipids as part of the bilayer membrane.The production of new fatty acids such as PUFAs may therefore create newcharacteristics of membrane functions within a cell or organism.

Cellular membranes serve a variety of functions in a cell. First andforemost, a membrane differentiates the contents of a cell from thesurrounding environment, thus giving integrity to the cell. Membranesmay also serve as barriers to the influx of hazardous or unwantedcompounds, and also to the efflux of desired compounds.

For more detailed descriptions and implications of membranes andinvolved mechanisms see: Bamberg, E. et al., (1993) Charge transport ofion pumps on lipid bilayer membranes, Q. Rev. Biophys. 26: 1-25; Gennis,R. B. (1989) Pores, Channels and Transporters, in: Biomembranes,Molecular Structure and Function, Springer: Heidelberg, p. 270-322; andNikaido, H. and Saier, H. (1992) Transport proteins in bacteria: commonthemes in their design, Science 258: 936-942, and references containedwithin each of these references.

Lipid synthesis may be divided into two parts: the synthesis of fattyacids and their attachment to sn-glycerol-3-phosphate, and the additionor modification of a polar head group. Typical lipids utilized inmembranes include phospholipids, glycolipids, sphingolipids, andphosphoglycerides. Fatty acid synthesis begins with the conversion ofacetyl CoA either to malonyl CoA by acetyl CoA carboxylase, or toacetyl-ACP by acetyltransacylase. Following a condensation reaction,these two product molecules together form further intermediates, whichare converted by a series of condensation, reduction and dehydrationreactions to yield a saturated fatty acid molecule having the desiredchain length. The production of unsaturated fatty acids from suchmolecules is catalyzed by specific desaturases either aerobically, withthe help of molecular oxygen, or anaerobically (for reference on fattyacid synthesis in microorganisms, see F. C. Neidhardt et al. (1996) E.coli and Salmonella. ASM Press: Washington, D.C., p. 612-636 andreferences contained therein; Lengeler et al. (eds) (1999) Biology ofProcaryotes. Thieme: Stuttgart, N.Y., and references contained therein;and Magnuson, K. et al., (1993) Microbiological Reviews 57: 522-542, andreferences contained therein).

Preferred precursors for the inventive PUFA biosynthesis process arelinoleic and linolenic acid. These C₁₈ carbon fatty acids have to beelongated to C₂₀ and C₂₂ in order to obtain eicosa and docosa chain typefatty acids. With the aid of various desaturases such as enzymes withΔ6- or Δ8- and Δ5- and Δ4-desaturases activity eicosapentaenoic acid anddocosahexaenoic acid as well as various other long chain PUFAs can beobtained, extracted and used for various purposes, for example in foodand feed applications.

For the production of long chain PUFAs it is necessary as mentionedabove that the polyunsaturated C₁₈ fatty acids are elongated by at leasttwo carbon atoms by the enzymatic activity of the inventive elongase.The nucleic acid sequence of the invention encodes the first plantelongase which has the ability to elongate α-linolenic acid(C_(18:3 d9, 12, 15)) by at least two carbon atoms but not γ-linolenicacid (C_(18:3 d6, 9, 12)).

Furthermore fatty acids have to be transported and incorporated into thetriacylglycerol storage lipid subsequent to various modifications.Another essential step in lipid synthesis is the transfer of fatty acidsonto the polar head groups by, for example,glycerol-phosphate-acyltransferase (see Frentzen, 1998, Lipid,100(4-5):161-166).

For publications on plant fatty acid biosynthesis, desaturation, lipidmetabolism and membrane transport of lipoic compounds, beta-oxidation,fatty acid modification and cofactors, triacylglycerol storage andassembly including references therein see following articles: Kinney,1997, Genetic Engineering, ed.: J K Setlow, 19:149-166; Ohlrogge andBrowse, 1995, Plant Cell 7:957-970; Shanklin and Cahoon, 1998, Annu.Rev. Plant Physiol. Plant Mol. Biol., 49:611-641; Voelker, 1996, GeneticEngineering, ed.: J K Setlow, 18:111-13; Gerhardt, 1992, Prog. Lipid R.31:397-417; Gühnemann-Schäfer & Kindl, 1995, Biochim. Biophys Acta1256:181-186; Kunau et al., 1995, Prog. Lipid Res. 34:267-342; Stymne etal 1993, in: Biochemistry and Molecular Biology of Membrane and StorrageLipids of Plants, Eds: Murata and Somerville, Rockville, AmericanSociety of Plant Physiologists, 150-158, Murphy & Ross 1998, PlantJournal. 13(1):1-16.

Vitamins, cofactors, and nutraceuticals such as PUFAs comprise a groupof molecules which the higher animals have lost the ability tosynthesize and so must ingest or which the higher animals cannotsufficiently produce on their own and so must ingest additionally,although they are readily synthesized by other organisms such asbacteria. The biosynthesis of these molecules in organisms capable ofproducing them, such as bacteria, has been largely characterized(Ullmann's Encyclopedia of Industrial Chemistry, Vitamins vol. A27, p.443-613, VCH: Weinheim, 1996; Michal, G. (1999) Biochemical Pathways: AnAtlas of Biochemistry and Molecular Biology, John Wiley & Sons; Ong, A.S., Niki, E. & Packer, L. (1995) Nutrition, Lipids, Health, and DiseaseProceedings of the UNESCO/Confederation of Scientific and TechnologicalAssociations in Malaysia, and the Society for Free Radical ResearchAsia, held Sep. 1-3, 1994 at Penang, Malaysia, AOCS Press: Champaign,Ill. X, 374 S).

These said molecules are either bioactive substances themselves, or areprecursors of biologically active substances which may serve as electroncarriers or intermediates in a variety of metabolic pathways. Aside fromtheir nutritive value, these compounds also have significant industrialvalue as coloring agents, antioxidants, and catalysts or otherprocessing aids. (For an overview of the structure, activity, andindustrial applications of these compounds, see, for example, Ullmann'sEncyclopedia of Industrial Chemistry, Vitamins vol. A27, p. 443-613,VCH: Weinheim, 1996.). Polyunsaturated fatty acids have variousfunctions and health benefit effects such as in coronary heart disease,inflammatory mechanisms, infant nutrition etc. For publications andreferences see including references cited therein: Simopoulos 1999, Am.J. Clin. Nutr., 70 (3 Suppl):560-569, Takahata et al., Biosc.Biotechnol. Biochem, 1998, 62 (11):2079-2085, Willich und Winther, 1995,Deutsche Medizinische Wochenschrift, 120 (7):229 ff.

II. Elements and Methods of the Invention

The present invention is based, at least in part, on the discovery ofnovel molecules, referred to herein as ASE nucleic acid and proteinmolecules, which have an effect on the production of cellular membranesin Isochrysis galbana and influence for example the movement ofmolecules across such membranes. In one embodiment, the ASE moleculesparticipate in the metabolism of compounds necessary for theconstruction of cellular membranes in microorganisms and plants, ordirectly influence the transport of molecules across these membranes. Ina preferred embodiment, the activity of the ASE molecules of the presentinvention to regulate membrane component production and membranetransport has an impact on the production of a desired fine chemical bythis organism. In a particularly preferred embodiment, the ASE moleculesof the invention are modulated in activity, such that themicroorganisms' or plants' metabolic pathways which the ASEs of theinvention regulate are modulated in yield, production, and/or efficiencyof production and the transport of compounds through the membranes isaltered in efficiency, which either directly or indirectly modulates theyield, production, and/or efficiency of production of a desired finechemical by microorganisms and plants.

The language, ASE or ASE polypeptide, includes proteins whichparticipate in the metabolism of compounds necessary for theconstruction of cellular membranes in microorganisms and plants, or inthe transport of molecules across these membranes. Examples of ASEs aredisclosed in SEQ ID NO: 1 or its derivatives. The terms ASE gene or ASEnucleic acid sequence include nucleic acid sequences encoding an ASE,which consist of a coding region and also corresponding untranslated 5′and 3′ sequence regions.

Examples of ASE genes include those set forth in SEQ ID NO: 1 and theirderivatives. The terms production or productivity are art-recognized andinclude the concentration of the fermentation product (for example, thedesired fine chemical) formed within a given time and a givenfermentation volume (e.g., kg product per hour per liter). The termefficiency of production includes the time required for a particularlevel of production to be achieved (for example, how long it takes forthe cell to attain a particular throughput of a fine chemical). The termyield or product/carbon yield is art-recognized and includes theefficiency of the conversion of the carbon source into the product(i.e., fine chemical). This is generally written as, for example, kgproduct per kg carbon source. By increasing the yield or production ofthe compound, the quantity of recovered molecules, or of usefulrecovered molecules, of that compound in a given amount of culture overa given amount of time is increased. The terms biosynthesis or abiosynthetic pathway are art-recognized and include the synthesis of acompound, preferably an organic compound, by a cell from intermediatecompounds in what may be a multistep and highly regulated process. Theterms degradation or a degradation pathway are art-recognized andinclude the breakdown of a compound, preferably an organic compound, bya cell to degradation products (generally speaking, smaller or lesscomplex molecules) in what may be a multistep and highly regulatedprocess. The language metabolism is art-recognized and includes thetotality of the biochemical reactions that take place in an organism.The metabolism of a particular compound, then, (e.g., the metabolism ofa fatty acid) comprises the overall biosynthetic, modification, anddegradation pathways in a cell related to this compound.

In another embodiment, the ASE molecules of the invention are capable ofmodulating the production of a desired molecule, such as a finechemical, in microorganisms or plants. There are a number of mechanismsby which the alteration of an ASE of the invention may directly affectthe yield, production, and/or efficiency of production of a finechemical from a microorganism or plant strain incorporating such analtered protein. Those ASEs involved in the transport of fine chemicalmolecules within or from the cell may be increased in number or activitysuch that greater quantities of these compounds are transported acrossmembranes, from which they are more readily recovered andinterconverted. Further, fatty acids and lipids themselves are desirablefine chemicals; by optimizing the activity or increasing the number ofone or more ASEs of the invention which participate in the biosynthesisof these compounds, or by impairing the activity of one or more ASEswhich are involved in the degradation of these compounds, it may bepossible to increase the yield, production, and/or efficiency ofproduction of fatty acid and lipid molecules from microorganisms orplants.

The mutagenesis of the ASE gene of the invention may also result in ASEshaving altered activities which indirectly impact on the production ofone or more desired fine chemicals from microorganisms or plants. Forexample, ASEs of the invention involved in the export of waste productsmay be increased in number or activity such that the normal metabolicwastes of the cell (possibly increased in quantity due to theoverproduction of the desired fine chemical) are efficiently exportedbefore they are able to damage molecules within the cell (which woulddecrease the viability of the cell) or to interfere with fine chemicalbiosynthetic pathways (which would decrease the yield, production, orefficiency of production of the desired fine chemical). Further, therelatively large intracellular quantities of the desired fine chemicalmay in themselves be toxic to the cell, so by increasing the activity ornumber of transporters able to export this compound from the cell, onemay increase the viability of the cell in culture, in turn leading to agreater number of cells in the culture producing the desired finechemical. The ASEs of the invention may also be manipulated such thatthe relative amounts of different lipid and fatty acid molecules areproduced. This may have a profound effect on the lipid composition ofthe membrane of the cell. Since each type of lipid has differentphysical properties, an alteration in the lipid composition of amembrane may significantly alter membrane fluidity. Changes in membranefluidity can impact the transport of molecules across the membrane, aswell as the integrity of the cell, both of which have a profound effecton the production of fine chemicals from microorganisms and plants inlarge-scale fermentative culture. Plant membranes confer specificcharacteristics such as tolerance towards heat, cold, salt, drought andtolerance towards pathogens like bacteria and fungi. Modulating membranecompounds therefore can have a profound effect on the plants' fitness tosurvive under aforementioned stress parameters. This can happen eithervia changes in signaling cascades or directly via the changed membranecomposition (for example see: Chapman, 1998, Trends in Plant Science, 3(11):419-426) and influence signaling cascades (see Wang 1999, PlantPhysiology, 120:645-651) or on cold tolerance as disclosed in WO95/18222.

The isolated nucleic acid sequence of the invention is contained withinthe genome of an Isochrysis galbana strain as described in the Examples.The nucleotide sequence of the isolated Isochrysis galbana ASE cDNA andthe predicted amino acid sequences of the Isochrysis galbana ASEs areshown in SEQ ID NO: 1 and 2, respectively.

A fragment of nucleic acid molecule in SEQ ID NO: 1 was isolated bypolymerase chain reaction with the aid of degenerated oligonucleotidesderived from other known elongase genes and a vector primer. A partialfragment was amplified further and used for the isolation of a fulllength cDNA containing sufficient sequence information representing afunctionally active ASE gene. One clone contained a complete ASE geneshowing weak homology to known elongase genes. The expression of theopen reading frame in yeast unforeseeingly revealed an ASE gene specificactivity. The enzyme elongates Δ9-fatty acids as shown in the Examples.

The present invention also pertains to proteins which have an amino acidsequence which is substantially homologous to an amino acid sequence ofSEQ ID NO: 2. As used herein, a protein which has an amino acid sequencewhich is substantially homologous to a selected amino acid sequence isat least about 50% homologous to the selected amino acid sequence, e.g.,the entire selected amino acid sequence. A protein which has an aminoacid sequence which is substantially homologous to a selected amino acidsequence can also be least about 50-60%, preferably at least about60-70%, and more preferably at least about 70-80%, 80-90%, or 90-95%,and most preferably at least about 96%, 97%, 98%, 99% or more homologousto the selected amino acid sequence.

The ASE of the invention or a biologically active portion or fragmentthereof can participate in the metabolism of compounds necessary for theconstruction of cellular membranes in microorganisms or plants, or inthe transport of molecules across these membranes, or have one or moreof the activities needed to elongate C18 PUFAs to yield in C₂₂ or C₂₄PUFAs as well as related PUFAs.

Various aspects of the invention are described in further detail in thefollowing subsections:

A. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode ASE polypeptides or biologically active portions thereof, aswell as nucleic acid fragments sufficient for use as hybridizationprobes or primers for the identification or amplification ofASE-encoding nucleic acid (e.g., ASE DNA). As used herein, the term“nucleic acid molecule” is intended to include DNA molecules (e.g., cDNAor genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA orRNA generated using nucleotide analogs. This term also encompassesuntranslated sequences located at both the 3′ and 5′ ends of the codingregion of the gene: at least about 500, preferably at least about 400,more preferably at least about 350, 300, 250, 200, 150 and even morepreferably at least about 100 nucleotides of sequence upstream from the5′ end of the coding region and at least about 1000, preferably at leastabout 500, more preferably at least about 400, 300, 250, 200, 150 andeven more preferably 100, 80, 60, 40 or 20 nucleotides of sequencedownstream from the 3′ end of the coding region of the gene. The nucleicacid molecule can be single-stranded or double-stranded, but preferablyis double-stranded DNA. An “isolated” nucleic acid molecule is one whichis separated from other nucleic acid molecules which are present in thenatural source of the nucleic acid. Preferably, an “isolated” nucleicacid is free of sequences which naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated ASE nucleic acid moleculecan contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1kb of nucleotide sequences which naturally flank the nucleic acidmolecule in genomic DNA of the cell from which the nucleic acid isderived (e.g, an Isochrysis galbana cell). Moreover, an “isolated”nucleic acid molecule, such as a cDNA molecule, can be substantiallyfree of other cellular material, or-culture medium when produced byrecombinant techniques, or chemical precursors or other chemicals whenchemically synthesized; the language “substantially free of cellularmaterial” includes preparations of nucleic acid molecules having lessthan about 30% (by dry weight) of other material such as proteins,polysaccharides etc. (also referred to herein as a “contaminatingmaterial”), more preferably less than about 20% of contaminatingmaterial, still more preferably less than about 10% of contaminatingmaterial, and most preferably less than about 5% of contaminatingmaterial.

One embodiment of the invention is an isolated nucleic acid derived froma plant encoding a polypeptide which elongates α-linolenic acid(C_(18:3 d9, 12, 15)) by at least two carbon atoms whereas γ-linolenicacid (C_(18:3 d6, 9, 12)) is not elongated.

A further embodiment of the invention is an isolated nucleic acidcomprising a nucleotide sequence which encodes a polypeptide whichelongates α-linolenic acid (C_(18:3 d9, 12, 15)) by at least two carbonatoms whereas γ-linolenic acid (C_(18:3 d6, 9, 12)) is not elongated,which nucleic acid is selected from the group consisting of

-   a) a nucleic acid sequence depicted in SEQ ID NO: 1,-   b) a nucleic acid sequence which encodes a polypeptide depicted in    SEQ ID NO: 2,-   c) derivatives of the sequence depicted in SEQ ID NO: 1, which    encodes polypeptides having at least 50% homology to the sequence    encoding amino acid sequences depicted in SEQ ID NO: 2 and which    sequences function as an elongase.

The abovementioned isolated nucleic acid of the invention is derivedfrom organisms such as ciliates, fungi, algae or dinoflagellates whichare able to synthesize PUFAs, preferably from plants, particularlypreferably from the genus Isochrysis and most particularly preferablyfrom Isochrysis galbana.

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode ASE polypeptides or biologically active portions thereof, aswell as nucleic acid fragments sufficient for use as hybridizationprobes or primers for the identification or amplification ofASE-encoding nucleic acid (e.g., ASE DNA). As used herein, the term“nucleic acid molecule” is intended to include DNA molecules (e.g., cDNAor genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA orRNA generated using nucleotide analogs. This term also encompasses theuntranslated sequence located at both the 3′ and 5′ ends of the codingregion of the gene: at least about 100 nucleotides of sequence upstreamfrom the 5′ end of the coding region and at least about 20 nucleotidesof sequence downstream from the 3′ end of the coding region of the gene.The nucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA. An “isolated” nucleic acid moleculeis one which is separated from other nucleic acid molecules which arepresent in the natural source of the nucleic acid. Preferably, an“isolated” nucleic acid is free of sequences which naturally flank thenucleic acid (i.e., sequences located at the 5′ and 3′ ends of thenucleic acid) in the genomic DNA of the organism from which the nucleicacid is derived. For example, in various embodiments, the isolated ASEnucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flankthe nucleic acid molecule in genomic DNA of the cell from which thenucleic acid is derived (e.g, an Isochrysis galbana cell). Moreover, an“isolated” nucleic acid molecule, such as a cDNA molecule, can besubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or chemical precursors or otherchemicals when chemically synthesized. Substantially free means that the[lacuna]

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having a nucleotide sequence of SEQ ID NO: 1, or a portionthereof, can be isolated using standard molecular biology techniques andthe sequence information provided herein. For example, an Isochrysisgalbana ASE cDNA can be isolated from an Isochrysis galbana libraryusing all or a portion of SEQ ID NO: 1 as a hybridization probe andstandard hybridization techniques (e.g., as described in Sambrook etal., Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold SpringHarbor Laboratory, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989). Moreover, a nucleic acid molecule encompassing allor a portion of one of the sequences of SEQ ID NO: 1 can be isolated bythe polymerase chain reaction using oligonucleotide primers designed onthe basis of this sequence or parts thereof, especially regions aroundhis-box motifs, see Shanklin et al. (1994) Biochemistry 33, 12787-12794(e.g., a nucleic acid molecule encompassing all or a portion of one ofthe sequences of SEQ ID NO: 1 can be isolated by the polymerase chainreaction using oligonucleotide primers designed on the basis of thissame sequence of SEQ ID NO: 1). For example, mRNA can be isolated fromplant cells (e.g., by the guanidinium-thiocyanate extraction procedureof Chirgwin et al. (1979) Biochemistry 18: 5294-5299) and cDNA can beprepared using reverse transcriptase (e.g., Moloney MLV reversetranscriptase, available from Gibco/BRL, Bethesda, Md.; or AMV reversetranscriptase, available from Seikagaku America, Inc., St. Petersburg,Fla.). Synthetic oligonucleotide primers for polymerase chain reactionamplification can be designed on the basis of one of the nucleotidesequence shown in SEQ ID NO: 1. A nucleic acid of the invention can beamplified using cDNA or, alternatively, genomic DNA as a template andappropriate oligonucleotide primers according to standard PCRamplification techniques. The nucleic acid so amplified can be clonedinto an appropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to an ASE nucleotidesequence can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

The cDNA shown in SEQUENCE ID NO: 1 comprises sequences encoding ASEs(i.e., the “coding region”), as well as 5′ untranslated sequence and 3′untranslated sequence information. Alternatively, the nucleic acidmolecule can comprise only the coding region of any of the sequences inSEQ ID NO: 1 or can contain whole genomic fragments isolated fromgenomic DNA.

The SEQUENCE ID NO: 2 is a translation of the coding region of thenucleotide sequence of nucleic acid molecule Ig_ASE1 shown in SEQ ID NO:1.

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which is a complement ofone of the nucleotide sequences shown in SEQ ID NO: 1, or a portionthereof. A nucleic acid molecule which is complementary to one of thenucleotide sequences shown in SEQ ID NO: 1 is one which is sufficientlycomplementary to one of the nucleotide sequences shown in SEQ ID NO: 1such that it can hybridize with one of the nucleotide sequences shown inSEQ ID NO: 1, thereby forming a stable duplex.

Homologs of the novel elongase nucleic acid sequence having the sequenceSEQ ID NO: 1 mean, for example, allelic variants which have at leastabout 50-60%, preferably at least about 60-70%, more preferably at leastabout 70-80%, 80-90%, or 90-95%, and even more preferably at least about95%, 96%, 97%, 98%, 99% or more homology to a nucleotide sequence shownin SEQ ID NO: 1 or its homologs, derivatives or analogs or portionsthereof. In an additional preferred embodiment, an isolated nucleic acidmolecule of the invention comprises a nucleotide sequence whichhybridizes, e.g., hybridizes under stringent conditions, with one of thenucleotide sequences shown in SEQ ID NO: 1, or a portion thereof.Allelic variants comprise, in particular, functional variants which areobtainable by deletion, insertion or substitution of nucleotides fromthe sequence depicted in SEQ ID NO: 1, the intention being, however,that the enzymatic activity of the derived synthesized proteinsadvantageously be retained for the insertion of one or more genes.Proteins which have still the enzymatic activity of the elongase meansproteins which have at least 10% of the original enzymatic activity,preferably 20%, particularly preferably 30%, most particularlypreferably 40%, in comparison to the protein encoded by SEQ ID NO: 2.

Homologs of SEQ ID NO: 1 additionally mean, for example, bacterial,fungal or plant homologs, truncated sequences, single-stranded DNA orRNA of the coding and noncoding DNA sequence.

Homologs of SEQ ID NO: 1 also mean derivatives such as, for example,promoter variants. The promoters upstream of the indicated nucleotidesequences may be modified by one or more nucleotide exchanges, byinsertion(s) and/or deletion(s) without, however, the functionality oractivity of the promoters being impaired. It is additionally possiblefor the promoters to have their activity increased by modifying theirsequence, or to be completely replaced by more active promoters evenfrom heterologous organisms.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the coding region of one of the sequences in SEQ ID NO: 1,for example a fragment which can be used as a probe or primer or afragment encoding a biologically active portion of an ASE. Thenucleotide sequences determined from the cloning of the ASE gene fromIsochrysis galbana allow for the generation of probes and primersdesigned for use in identifying and/or cloning ASE homologs in othercell types and organisms, as well as ASE homologs from Isochrysisgalbana or related species. The probe/primer typically comprisessubstantially purified oligonucleotide. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions with at least about 12, preferably about 16, morepreferably about 25, 40, 50 or 75 consecutive nucleotides of a sensestrand of one of the sequences set forth in SEQUENCE ID NO: 1, anantisense sequence of one of the sequences set forth in sequence ID NO:1, or naturally occurring mutants thereof. Primers based on a nucleotidesequence of SEQUENCE ID NO: 1 can be used in PCR reactions to clone ASEhomologs. Probes based on the ASE nucleotide sequences can be used todetect transcripts or genomic sequences encoding the same or homologousproteins. In preferred embodiments, the probe further comprises a labelgroup attached thereto, e.g. the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as a part of a genomic marker test kit for identifying cellswhich misexpress an ASE, such as by measuring a level of an ASE-encodingnucleic acid in a sample of cells, e.g., detecting ASE mRNA levels ordetermining whether a genomic ASE gene has been mutated or deleted.

In one embodiment, the nucleic acid molecule of the invention encodes aprotein or portion thereof which includes an amino acid sequence whichis sufficiently homologous to an amino acid sequence of SEQ ID NO: 2such that the protein or portion thereof maintains the ability toparticipate in the metabolism of compounds necessary for theconstruction of cellular membranes in microorganisms or plants, or inthe transport of molecules across these membranes. As used herein, theterm “sufficiently homologous” refers to proteins or portions thereofwhich have amino acid sequences which include a minimum number ofidentical or equivalent (e.g., an amino acid residue which has a similarside chain as an amino acid residue in one of the sequences of SEQ IDNO: 2) amino acid residues to an amino acid sequence of SEQ ID NO: 2such that the protein or portion thereof is able to participate in themetabolism of compounds necessary for the construction of cellularmembranes in microorganisms or plants, or in the transport of moleculesacross these membranes. Protein members of such membrane componentmetabolic pathways or membrane transport systems, as described herein,may play a role in the production and secretion of one or more finechemicals. Examples of such activities are also described herein. Thus,the function of an ASE contributes either directly or indirectly to theyield, production, and/or efficiency of production of one or more finechemicals. Examples of ASE substrate specificities of the catalyticactivity are set forth in Tab. 2.

In another embodiment, derivatives of the nucleic acid molecule of theinvention encode proteins which are at least about 50-60%, preferably atleast about 60-70%, and more preferably at least about 70-80%, 80-90%,90-95%, and most preferably at least about 96%, 97%, 98%, 99% or morehomologous to an entire amino acid sequence of SEQ ID NO: 2.

Portions of proteins encoded by the ASE nucleic acid molecules of theinvention are preferably biologically active portions of one of theASEs. As used herein, the term “biologically active portion of an ASE”is intended to include a portion, e.g., a domain/motif, of an ASE thatparticipates in the metabolism of compounds necessary for theconstruction of cellular membranes in microorganisms or plants, or inthe transport of molecules across these membranes, or has an activity asset forth in Tab. 2. To determine whether an ASE or a biologicallyactive portion thereof can participate in the metabolism of compoundsnecessary for the construction of cellular membranes in microorganismsor plants, or in the transport of molecules across these membranes, anassay of enzymatic activity may be performed. Such assay methods arewell known to those skilled in the art, as detailed in Example 8 of theExamples.

Additional nucleic acid fragments encoding biologically active portionsof an ASE can be prepared by isolating a portion of one of the sequencesin SEQ ID NO: 2, expressing the encoded portion of the ASE or peptide(e.g., by recombinant expression in vitro) and assessing the activity ofthe encoded portion of the ASE or peptide.

The invention further encompasses nucleic acid molecules that differfrom one of the nucleotide sequences shown in SEQ ID NO: 1 (and portionsthereof) due to degeneracy of the genetic code and thus encode the sameASE as that encoded by the nucleotide sequences shown in SEQ ID NO: 1.In another embodiment, an isolated nucleic acid molecule of theinvention has a nucleotide sequence encoding a protein having an aminoacid sequence shown in SEQ ID NO: 2. In a further embodiment, thenucleic acid molecule of the invention encodes a full length Isochrysisgalbana protein which is substantially homologous to an amino acidsequence of SEQ ID NO: 2 (encoded by an open reading frame shown in SEQID NO: 1).

In addition to the Isochrysis galbana ASE nucleotide sequences shown inSEQ ID NO: 1, it will be appreciated by those skilled in the art thatDNA sequence polymorphisms that lead to changes in the amino acidsequences of ASEs may exist within a population (e.g., the Isochrysisgalbana population). Such genetic polymorphisms in the ASE gene mayexist among individuals within a population due to natural variation. Asused herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules comprising an open reading frame encoding an ASE,preferably an Isochrysis galbana ASE. Such natural variations cantypically result in 1-5% variance in the nucleotide sequence of the ASEgene. Any and all such nucleotide variations and resulting amino acidpolymorphisms in ASE that are the result of natural variation and thatdo not alter the functional activity of ASEs are intended to be withinthe scope of the invention.

Nucleic acid molecules corresponding to natural variants andnon-Isochrysis galbana homologs of the Isochrysis galbana ASE cDNA ofthe invention can be isolated based on their homology to Isochrysisgalbana ASE nucleic acid disclosed herein using the Isochrysis galbanacDNA, or a portion thereof, as a hybridization probe according tostandard hybridization techniques under stringent hybridizationconditions. Accordingly, in another embodiment, an isolated nucleic acidmolecule of the invention is at least 15 nucleotides in length andhybridizes under stringent conditions to the nucleic acid moleculecomprising a nucleotide sequence of SEQ ID NO: 1. In other embodiments,the nucleic acid is at least 25, 50, 100, 250 or more nucleotides inlength. As used herein, the term “hybridizes under stringent conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 60% homologous to each othertypically remain hybridized to each other. Preferably, the conditionsare such that sequences at least about 65%, more preferably at leastabout 70%, and even more preferably at least about 75% or morehomologous to each other typically remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, John Wiley & Sons, NewYork (1989), 6.3.1-6.3.6. A preferred, nonlimiting example of stringenthybridization conditions is hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45 degree C., followed by one or more washes in0.2×SSC, 0.1% SDS at 50-65 degree C. Preferably, an isolated nucleicacid molecule of the invention that hybridizes under stringentconditions to a sequence of SEQ ID NO: 1 corresponds to anaturally-occurring nucleic acid molecule. As used herein, a“naturally-occurring” nucleic acid molecule refers to an RNA or DNAmolecule having a nucleotide sequence that occurs in nature, (e.g.,encodes a natural protein). In one embodiment, the nucleic acid encodesa natural Isochrysis galbana ASE.

In addition to naturally-occurring variants of the ASE sequence that mayexist in the population, the skilled artisan will further appreciatethat changes can be introduced by mutation into a nucleotide sequence ofSEQ ID NO: 1, thereby leading to changes in the amino acid sequence ofthe encoded ASE, without altering the functional ability of the ASE. Forexample, nucleotide substitutions leading to amino acid substitutions at“nonessential” amino acid residues can be made in a sequence of SEQ IDNO: 1. A “nonessential” amino acid residue is a residue that can bealtered from a wild-type sequence of one of the ASEs (SEQ ID NO: 2)without altering the activity of said ASE, whereas an “essential” aminoacid residue is required for ASE activity. Other amino acid residues,however, (e.g., those that are not conserved or only semiconserved inthe domain having ASE activity) may not be essential for activity andthus are likely to be amenable to alteration without altering ASEactivity.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding ASEs that contain modified amino acid residues thatare not essential for ASE activity. Such ASEs differ in amino acidsequence from a sequence contained in SEQ ID NO: 2 yet retain at leastone of the ASE activities described herein. In one embodiment, theisolated nucleic acid molecule comprises a nucleotide sequence encodinga protein, wherein the protein comprises an amino acid sequence at leastabout 50% homologous to an amino acid sequence of SEQ ID NO: 2 and iscapable of participation in the metabolism of compounds necessary forthe construction of cellular membranes in Isochrysis galbana, or in thetransport of molecules across these membranes, or has one or moreactivities set forth in Tab. 2. Preferably, the protein encoded by thenucleic acid molecule is at least about 50-60% homologous to one of thesequences in SEQUENCE ID NO: 2, more preferably at least about 60-70%homologous to one of the sequences in SEQ ID NO: 2, even more preferablyat least about 70-80%, 80-90%, 90-95% homologous to one of the sequencesin SEQUENCE ID NO: 2, and most preferably at least about 96%, 97%, 98%,or 99% homologous to one of the sequences in SEQ ID NO: 2.

To determine the percent homology of two amino acid sequences (e.g., oneof the sequences of SEQ ID NO: 2 and a mutant form thereof) or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of one protein or nucleicacid for optimal alignment with the other protein or nucleic acid). Theamino acid residues or nucleotides at corresponding amino acid positionsor nucleotide positions are then compared. When a position in onesequence (e.g., one of the sequences of SEQ ID NO: 2) is occupied by thesame amino acid residue or nucleotide as the corresponding position inthe other sequence (e.g., a mutant form of the form selected from SEQ IDNO: 2), then the molecules are homologous at that position (i.e., asused herein amino acid or nucleic acid “homology” is equivalent to aminoacid or nucleic acid “identity”). The percent homology between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % homology=numbers of identical positions/totalnumbers of positions×100).

An isolated nucleic acid molecule encoding an ASE homologous to aprotein sequence of SEQ ID NO: 2 can be created by introducing one ormore nucleotide substitutions, additions or deletions into a nucleotidesequence of SEQ ID NO: 1 such that one or more amino acid substitutions,additions or deletions are introduced into the encoded protein.Mutations can be introduced into one of the sequences of SEQ ID NO: 1 orits derivatives by standard techniques, such as site-directedmutagenesis and PCR-mediated mutagenesis. Preferably, conservative aminoacid substitutions are made at one or more predicted nonessential aminoacid residues. A “conservative amino acid substitution” is one in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a predicted nonessential amino acid residue in an ASEis preferably replaced with another amino acid residue from the sameside chain family. Alternatively, in another embodiment, mutations canbe introduced randomly along all or part of the ASE coding sequence,such as by saturation mutagenesis, and the resultant mutants can bescreened for an ASE activity described herein to identify mutants thatretain ASE activity. Following mutagenesis of one of the sequences ofSEQ ID NO: 1, the encoded protein can be expressed recombinantly and theactivity of the protein can be determined using, for example, assaysdescribed herein (see Examples).

A further known technique for directed evolution and mutagenesis of genesequences encoding enzymes is gene shuffling (Stemmer, PNAS 1994, 91:10747-10751, WO 97/20078 and WO 98/13487). Gene shuffling is a methodfor the combination of gene fragments and can be combined with errorprone PCR in order to further enhance the genetic variability ofresulting sequences and encoded enzymatic activities. A premise for suchan approach is a suitable screening system. In the case of elongaseshigh throughput metabolite measurements facilitated by MALDI-TOF, gaschromatography-mass spectroscopy, thin layer chromatography or liquidchromatography-mass spectroscopy or other suitable combinations ormethods can be used to monitor the appearance of new compounds orproducts in the hydrophobic fraction.

In addition to the nucleic acid molecules encoding ASEs described above,another aspect of the invention pertains to isolated nucleic acidmolecules which are antisense to an isolated nucleic acid comprising anucleotide sequence which encodes a polypeptide which elongatesα-linolenic acid (C_(18:3 d9, 12, 15)) by at least two carbon atomswhereas γ-linolenic acid (C_(18:3 d6, 9, 12)) is not elongated, selectedfrom the group consisting of

-   a) a nucleic acid sequence depicted in SEQ ID NO: 1,-   b) a nucleic acid sequence which encodes a polypeptide depicted in    SEQ ID NO: 2,-   c) derivatives of the sequence depicted in SEQ ID NO: 1, which    encodes polypeptides having at least 50% homology to the sequence    encoding amino acid sequences depicted in SEQ ID NO: 2 and which    sequences function as an elongase.

An “antisense” nucleic acid comprises a nucleotide sequence which iscomplementary to a “sense” nucleic acid encoding a protein, e.g.,complementary to the coding strand of a double-stranded cDNA molecule orcomplementary to an mRNA sequence. Accordingly, an antisense nucleicacid can form hydrogen bonds with a sense nucleic acid. The antisensenucleic acid can be complementary to an entire ASE coding strand, or toonly a portion thereof. In one embodiment, an antisense nucleic acidmolecule is antisense to a “coding region” of the coding strand of anucleotide sequence encoding an ASE. The term “coding region” refers tothe region of the nucleotide sequence comprising codons which aretranslated into amino acid residues (e.g., the entire coding regionstarting with and ending with the stop codon, i.e. the last codon beforethe stop codon). In another embodiment, the antisense nucleic acidmolecule is antisense to a “noncoding region” of the coding strand of anucleotide sequence encoding ASE. The term “noncoding region” refers to5′ and 3′ sequences which flank the coding region that are nottranslated into amino acids (i.e., also referred to as 5′ and 3′untranslated regions). It is also possible to use the inverted repeattechnology combining an antisense fragment with a portion of theantisense fragment in sense orientation linked by either an adaptersequence or an excisable intron (Abstract Book of the 6^(th) Intern.Congr. Of Plant Mol Biol. ISPMB, Quebec Jun. 18-24, 2000, Abstract No.S20-9 by Green et al.).

Given the coding strand sequences encoding the ASE disclosed herein(e.g., the sequences set forth in SEQ ID NO: 1), antisense nucleic acidsof the invention can be designed according to the rules of Watson andCrick base pairing. The antisense nucleic acid molecule can becomplementary to the entire coding region of ASE mRNA, but morepreferably is an oligonucleotide which is antisense to only a portion ofthe coding or noncoding region of ASE mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of ASE mRNA. An antisense oligonucleotide can be,for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 and morenucleotides in length. An antisense nucleic acid of the invention can beconstructed using chemical synthesis and enzymatic ligation reactionsusing procedures known in the art. For example, an antisense nucleicacid (e.g., an antisense oligonucleotide) can be chemically synthesizedusing naturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between theantisense and sense nucleic acids, e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used. Examples of modifiednucleotides which can be used to generate the antisense nucleic acidinclude 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a cell or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding an ASE tothereby inhibit expression of the protein, e.g., by inhibitingtranscription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. The antisense molecule can be modified such that itspecifically binds to a receptor or an antigen expressed on the selectedcell surface, e.g., by linking the antisense nucleic acid molecule to apeptide or an antibody which binds to a cell surface receptor orantigen. The antisense nucleic acid molecule can also be delivered tocells using the vectors described below. To achieve sufficientintracellular concentrations of the antisense molecules, vectorconstructs in which the antisense nucleic acid molecule is placed underthe control of a strong prokaryotic, viral, or eukaryotic promoter,including plant promoters, are preferred.

In another embodiment, the antisense nucleic acid molecule of theinvention is an anomeric nucleic acid molecule. An anomeric nucleic acidmolecule forms specific double-stranded hybrids with complementary RNAin which, contrary to the usual units, the strands run parallel to eachother (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). Theantisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analog (Inoue et al. (1987) FEBSLett. 215:327-330).

In another embodiment, an antisense nucleic acid of the invention is aribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity which are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes (described in Haselhoff andGerlach (1988) Nature 334:585-591)) can be used to catalytically cleaveASE mRNA transcripts to thereby inhibit translation of ASE mRNA. Aribozyme having specificity for an ASE-encoding nucleic acid can bedesigned on the basis of the nucleotide sequence of an ASE cDNAdisclosed herein in SEQ ID NO: 1 or on the basis of a heterologoussequence to be isolated according to methods taught in this invention.For example, a derivative of a Tetrahymena L-19 IVS RNA can beconstructed in which the nucleotide sequence of the active site iscomplementary to the nucleotide sequence to be cleaved in anASE-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071 andCech et al. U.S. Pat. No. 5,116,742. Alternatively, ASE mRNA can be usedto select a catalytic RNA having a specific ribonuclease activity from apool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W. (1993)Science 261:1411-1418.

Alternatively, ASE gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of an ASEnucleotide sequence (e.g., an ASE promoter and/or enhancers) to formtriple helical structures that prevent transcription of an ASE gene intarget cells. See generally Helene, C. (1991) Anticancer Drug Res.6(6):569-84; Helene, C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36;and Maher, L. J. (1992) Bioassays 14(12):807-15.

B. Gene Construct

Another embodiment of the invention is a novel gene construct comprisingan isolated nucleic acid derived from a plant which encodes apolypeptide which elongates α-linolenic acid (C_(18:3 d9, 12, 15)) by atleast two carbon atoms but not γ-linolenic acid (C_(18:3 d6, 9, 12)), orthe gene sequence of SEQ ID NO: 1, its homologs, derivatives or analogsas defined above which have been functionally linked to one or moreregulatory signals, advantageously to increase gene expression. Examplesof these regulatory sequences are sequences to which inducers orrepressors bind and thus regulate the expression of the nucleic acid. Inaddition to these novel regulatory sequences, the natural regulation ofthese sequences in front of the actual structural genes can still bepresent and, where appropriate, have been genetically modified so thatthe natural regulation has been switched off and the expression of thegenes has been increased. The gene construct can, however, also have asimpler structure, that is to say no additional regulatory signals havebeen inserted in front of the sequence SEQ ID NO: 1 or its homologs, andthe natural promoter with its regulation has not been deleted. Instead,the natural regulatory sequence has been mutated so that regulation nolonger takes place, and gene expression is enhanced. The gene constructmay additionally advantageously comprise one or more so-called enhancersequences functionally linked to the promoter and making increasedexpression of the nucleic acid sequence possible. It is also possible toinsert at the 3′ end of the DNA sequences additional advantageoussequences, such as further regulatory elements or terminators. Theelongase genes may be present in one or more copies in the geneconstruct. It is advantageous for further genes to be present in thegene construct for insertion of further genes into organisms.

Advantageous regulatory sequences for the novel process are present, forexample, in promoters such as cos-, tac-, trp-, tet-, trp-tet-, lpp-,lac-, lpp-lac-, lacI^(q-), T7-, T5-, T3-, gal-, trc-, ara-, SP6-,λ-P_(R)- or λ-P_(L)-promoter and are advantageously used inGram-negative bacteria. Further advantageous regulatory sequences arepresent, for example, in the Gram-positive promoters amy and SP02, inthe yeast or fungal promoters ADC1, MFα, AC, P-60, CYC1, GAPDH, TEF,rp28, ADH or in the plant promoters CaMV/35S [Franck et al., Cell 21(1980) 285-294], PRP1 [Ward et al., Plant. Mol. Biol. 22 (1993)], SSU,OCS, lib4, usp, STLS1, B33, nos or in the ubiquitin or phaseolinpromoter. Also advantageous in this connection are inducible promoterssuch as the promoters described in EP-A-0 388 186(benzenesulfonamide-inducible), Plant J. 2, 1992: 397-404 (Gatz et al.,tetracyclin-inducible), EP-A-0 335 528 (abscisic acid-inducible) or WO93/21334 (ethanol or cyclohexenol inducible). Additional useful plantpromoters are the cytosolic FBPase promoter or ST-LSI promoter of thepotato (Stockhaus et al., EMBO J. 8, 1989, 2445), thephosphoribosylpyrophoshate amidotransferase promoter of Glycine max(gene bank accession No. U87999) or the node-specific promoter describedin EP-A-0 249 676. Particularly advantageous promoters are promoterswhich allow the expression in tissues which are involved in the fattyacid biosynthesis. Most particularly advantageous are seed specificpromoters such as usp-, LEB4-, phaseolin or napin promoter. Additionalparticularly advantageous promoters are seed-specific promoters whichcan be used for monocots or dicots and which are described in U.S. Pat.No. 5,608,152 (napin promoter from rapeseed), WO 98/45461 (phaseolinpromoter from Arabidopsis), U.S. Pat. No. 5,504,200 (phaseolin promoterfrom Phaseolus vulgaris), WO 91/13980 (Bce4 promoter from Brassica),Baeumlein et al., Plant J., 2, 2, 1992: 233-239 (LEB4 promoter fromlegumes); said promoters are useful in dicots. The following promotersare useful for example in monocotyledons: lpt-2- or lpt-1-promoter frombarley (WO 95/15389 and WO 95/23230), hordein promoter from barley andother useful promoters described in WO 99/16890.

It is possible in principle to use all natural promoters with theirregulatory sequences like those mentioned above for the novel process.It is also possible and advantageous in addition to use syntheticpromoters.

The gene construct may, as described above, also comprise further geneswhich are to be inserted into the organisms. It is possible andadvantageous to insert and express in host organisms regulatory genessuch as genes for inducers, repressors or enzymes which intervene bytheir enzymatic activity in the regulation, or one or more or all genesof a biosynthetic pathway. These genes can be heterologous or homologousin origin. The inserted genes may have their own promoter or else beunder the control of the promoter of the sequence SEQ ID NO: 1 or itshomologs.

The gene construct advantageously comprises, for expression of the othergenes present, additional 3′ and/or 5′ terminal regulatory sequences toenhance expression, which are selected for optimal expression dependingon the selected host organism and gene or genes.

These regulatory sequences are intended to make specific expression ofthe genes and protein expression possible as mentioned above. This maymean, depending on the host organism, for example that the gene isexpressed or overexpressed only after induction, or that it isimmediately expressed and/or overexpressed.

The regulatory sequences or factors may moreover preferably have abeneficial effect on expression of the introduced genes, and thusincrease it. It is possible in this way for the regulatory elements tobe enhanced advantageously at the transcription level by using strongtranscription signals such as promoters and/or enhancers. However, inaddition, it is also possible to enhance translation by, for example,improving the stability of the mRNA.

In addition the inventive gene construct preferably comprises additionalgenes of different biochemical pathways, for example genes for thesynthesis of vitamins, carotinoids, sugars such as monosaccharides,oligosaccharides or polysaccharides, or fatty acid biosynthesis genes,more preferably the gene construct comprises fatty acid biosynthesisgenes such as desaturases, hydroxylases, Acyl-ACP-thioesterases,elongases, acetylenases, synthesases or reductases such as Δ19-, Δ17-,Δ15-, Δ12-, Δ9-, Δ8-, Δ6-, Δ5-, Δ4-desaturases, hydroxylases, elongases,Δ12-acetylenase, Acyl-ACP-thioesterasen, β-ketoacyl-ACP-synthases orβ-ketoacyl-ACP-reductases. Preferably the gene construct comprises fattyacid biosynthesis genes selected from the group consisting of Δ19-,Δ17-, Δ15-, Δ12-, Δ9-, Δ8-, Δ6-, Δ5-, Δ4-desaturases, hydroxylases,elongases, Δ12-acetylenase, acyl-ACP-thioesterases,β-ketoacyl-ACP-synthases or β-ketoacyl-ACP-reductases.

C. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding an ASE (or aportion thereof). As used herein, the term “vector” refers to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they havebeen introduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,nonepisomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise at leastone inventive nucleic acid or at least one inventive gene construct ofthe invention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vectors includeone or more regulatory sequences, selected on the basis of the hostcells used for expression, which is or are linked operably to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “linked operably” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence and thesesequences are fused to each other so that both sequences fulfill theproposed function ascribed to the sequence used (e.g., in an in vitrotranscription/translation system or in a host cell when the vector isintroduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990) or see:Gruber and Crosby, in: Methods in Plant Molecular Biology andBiotechnolgy, CRC Press, Boca Raton, Fla., eds.: Glick and Thompson,Chapter 7, 89-108 including the references therein. Regulatory sequencesinclude those which govern constitutive expression of a nucleotidesequence in many types of host cell and those which govern directexpression of the nucleotide sequence only in certain host cells undercertain conditions. It will be appreciated by those skilled in the artthat the design of the expression vector can depend on such factors asthe choice of the host cell to be transformed, the level of expressionof protein desired, etc. The expression vectors of the invention can beintroduced into host cells to thereby produce proteins or peptides,including fusion proteins or peptides, encoded by nucleic acids asdescribed herein (e.g., ASEs, mutant forms of ASEs, fusion proteins,etc.).

The recombinant expression vectors of the invention can be designed forexpression of ASEs in prokaryotic or eukaryotic cells. For example, ASEgenes can be expressed in bacterial cells such as C. glutamicum, insectcells (using baculovirus expression vectors), yeast and other fungalcells (see Romanos, M. A. et al. (1992) Foreign gene expression inyeast: a review, Yeast 8: 423-488; van den Hondel, C. A. M. J. J. et al.(1991) Heterologous gene expression in filamentous fungi, in: More GeneManipulations in Fungi, J. W. Bennet & L. L. Lasure, eds., p. 396-428:Academic Press: San Diego; and van den Hondel, C. A. M. J. J. & Punt, P.J. (1991) Gene transfer systems and vector development for filamentousfungi, in: Applied Molecular Genetics of Fungi, Peberdy, J. F. et al.,eds., p. 1-28, Cambridge University Press: Cambridge), algae (Falciatoreet al., 1999, Marine Biotechnology. 1, 3:239-251), ciliates of thetypes: Holotrichia, Peritrichia, Spirotrichia, Suctoria, Tetrahymena,Paramecium, Colpidium, Glaucoma, Platyophrya, Potomacus,Pseudocohnilembus, Euplotes, Engelmaniella, and Stylonychia, especiallyof the genus Stylonychia lemnae, with vectors following a transformationmethod as described in WO9801572, and multicellular plant cells (seeSchmidt, R. and Willmitzer, L. (1988), High efficiency Agrobacteriumtumefaciens-mediated transformation of Arabidopsis thaliana leaf andcotyledon explants, Plant Cell Rep.: 583-586); Plant Molecular Biologyand Biotechnology, C Press, Boca Raton, Fla., chapter 6/7, p. 71-119(1993); F. F. White, B. Jenes et al., Techniques for Gene Transfer, in:Transgenic Plants, Vol. 1, Engineering and Utilization, eds.: Kung undR. Wu, Academic Press (1993), 128-43; Potrykus, Annu. Rev. PlantPhysiol. Plant Molec. Biol. 42 (1991), 205-225 (and references citedtherein) or mammalian cells. Suitable host cells are discussed furtherin Goeddel, Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1990). Alternatively, the recombinantexpression vector can be transcribed and translated in vitro, forexample using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out withvectors containing constitutive or inducible promoters directing theexpression of either fusion or nonfusion proteins. Fusion vectors add anumber of amino acids to a protein encoded therein, usually to the aminoterminus of the recombinant protein but also to the C-terminus or fusedwithin suitable regions in the proteins. Such fusion vectors typicallyserve three purposes: 1) to increase expression of recombinant protein;2) to increase the solubility of the recombinant protein; and 3) to aidin the purification of the recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase.

Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein. In oneembodiment, the coding sequence of the elongase ASE is cloned into apGEX expression vector to create a vector encoding a fusion proteincomprising, from the N-terminus to the C-terminus, GST thrombin cleavagesite X protein. The fusion protein can be purified by affinitychromatography using glutathione-agarose resin. Recombinant ASE unfusedto GST can be recovered by cleavage of the fusion protein with thrombin.

Examples of suitable inducible nonfusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studieret al., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89). Target gene expression from thepTrc vector relies on host RNA polymerase transcription from a hybridtrp-lac fusion promoter. Target gene expression from the pET 11d vectorrelies on transcription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a resident λprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

Other vectors which are useful in prokaryotic organisms are known to aperson skilled in the art; such vectors are for example in E. colipLG338, pACYC184, the pBR series such as pBR322, the pUC series such aspUC18 or pUC19, the M113mp series, pKC30, pRep4, pHS1, pHS2, pPLc236,pMBL24, pLG200, pUR290, pIN-III¹¹³-B1, λgt11 or pBdCI, in StreptomycespIJ101, pIJ364, pIJ702 or pIJ361, in Bacillus pUB110, pC194 or pBD214,in Corynebacterium pSA77 or pAJ667.

One strategy to maximize recombinant protein expression is to expressthe protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in the bacterium chosen for expression, such asC. glutamicum (Wada et al. (1992) Nucleic Acids Res. 20:2111-2118). Suchalteration of nucleic acid sequences of the invention can be carried outby standard DNA synthesis techniques.

In another embodiment, the ASE expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1 (Baldari, et al., (1987) Embo J. 6:229-234), pMFa(Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al.,(1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego,Calif.). Vectors and methods for the construction of vectors appropriatefor use in other fungi, such as the filamentous fungi, include thosedetailed in: van den Hondel, C. A. M. J. J. & Punt, P. J. (1991) “Genetransfer systems and vector development for filamentous fungi, in:Applied Molecular Genetics of Fungi, J. F. Peberdy, et al., eds., p.1-28, Cambridge University Press: Cambridge or in: More GeneManipulations in Fungi [J. W. Bennet & L. L. Lasure, eds., p. 396-428:Academic Press: San Diego]. Additional useful yeast vectors are forexample 2 μM, pAG-1, YEp6, YEp13 or pEMBLYe23.

Alternatively, the ASEs of the invention can be expressed in insectcells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol.3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology170:31-39).

The abovementioned vectors are only a small overview of possible usefulvectors. Additional plasmids are well known by the skilled artisan andare described for example in: Cloning Vectors (Eds. Pouwels P. H. et al.Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018).

In another embodiment, a nucleic acid of the invention is expressed inmammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, B. (1987) Nature329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When usedin mammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyomavirus, Adenovirus 2, cytomegalovirusand Simian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Nonlimiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43:235-275), in particular promoters of Tcell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) PNAS 86:5473-5477),pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Patent Application Publication No.264,166). Developmentally-regulated promoters are also encompassed, forexample the murine hox promoters (Kessel and Gruss (1990) Science249:374-379) and the fetoprotein promoter (Campes and Tilghman (1989)Genes Dev. 3:537-546).

In another embodiment, the ASEs of the invention may be expressed inunicellular plant cells (such as algae), see Falciatore et al., 1999,Marine Biotechnology. 1 (3):239-251 and references therein, and plantcells from higher plants (e.g., the spermatophytes, such as cropplants). Examples of plant expression vectors include those detailed in:Becker, D., Kemper, E., Schell, J. and Masterson, R. (1992) “New plantbinary vectors with selectable markers located proximal to the leftborder”, Plant Mol. Biol. 20: 1195-1197; and Bevan, M. W. (1984) “BinaryAgrobacterium vectors for plant transformation, Nucl. Acid. Res. 12:8711-8721; Vectors for Gene Transfer in Higher Plants; in: TransgenicPlants, Vol. 1, Engineering and Utilization, eds.: Kung und R. Wu,Academic Press, 1993, p. 15-38.

A plant expression cassette preferably contains regulatory sequencescapable of driving gene expression in plants cells and which are linkedoperably so that each sequence can fulfill its function such astermination of transcription, such as polyadenylation signals. Preferredpolyadenylation signals are those originating from Agrobacteriumtumefaciens T-DNA such as the gene 3 known as octopine synthase of theTi-plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984), 835 ff) orfunctional equivalents thereof but also all other terminatorsfunctionally active in plants are suitable.

As plant gene expression is very often not limited on transcriptionallevels a plant expression cassette preferably contains other operablylinked sequences like translational enhancers such as the overdrivesequence containing the 5′-untranslated leader sequence from tobaccomosaic virus enhancing the protein per RNA ratio (Gallie et al 1987,Nucl. Acids Research 15:8693-8711).

Plant gene expression has to be linked operably to an appropriatepromoter conferring gene expression in a time, cell or tissue specificmanner. Preferred are promoters driving constitutive expression (Benfeyet al., EMBO J. 8 (1989) 2195-2202) like those derived from plantviruses like the 35S CaMV (Franck et al., Cell 21 1980) 285-294), the19S CaMV (see also U.S. Pat. No. 5,352,605 and WO 84/02913) or plantpromoters like those from Rubisco small subunit described in U.S. Pat.No. 4,962,028. Additionally vATPase-gene promoters such as a 1153basepair fragment from Beta vulgaris (Plant Mol Biol, 1999, 39:463-475)can be used to drive ASE gene expression alone or in combination withother PUFA biosynthesis genes.

Other preferred sequences for use in operable linkage in plant geneexpression cassettes are targeting sequences necessary to direct thegene product in its appropriate cell compartment (for review seeKermode, Crit. Rev. Plant Sci. 15, 4 (1996), 285-423 and referencescited therein) such as the vacuole, the nucleus, all types of plastidslike amyloplasts, chloroplasts, chromoplasts, the extracellular space,mitochondria, the endoplasmic reticulum, oil bodies, peroxisomes andother compartments of plant cells.

Plant gene expression can also be facilitated via a chemically induciblepromoter (for review see Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol.Biol., 48:89-108). Chemically inducible promoters are especiallysuitable if gene expression is wanted to occur in a time specificmanner. Examples of such promoters are a salicylic acid induciblepromoter (WO 95/19443), a tetracycline inducible promoter (Gatz et al.,(1992) Plant J. 2, 397-404) and an ethanol inducible promoter (WO93/21334).

Also promoters responding to biotic or abiotic stress conditions aresuitable promoters such as the pathogen inducible PRP1 gene promoter(Ward et al., Plant. Mol. Biol. 22 (1993), 361-366), the heat induciblehsp80 promoter from tomato (U.S. Pat. No. 5,187,267), cold induciblealpha-amylase promoter from potato (WO 96/12814) or the wound-induciblepinII promoter (EP-A-0 375 091).

Especially those promoters are preferred which confer gene expression intissues and organs where lipid and oil biosynthesis occurs, in seedcells such as cells of the endosperm and the developing embryo. Suitablepromoters are the napin-gene promoter from rapeseed (U.S. Pat. No.5,608,152), the USP-promoter from Vicia faba (Baeumlein et al., Mol GenGenet, 1991, 225 (3):459-67), the oleosin-promoter from Arabidopsis (WO98/45461), the phaseolin-promoter from Phaseolus vulgaris (U.S. Pat. No.5,504,200), the Bce4-promoter from Brassica (WO 91/13980) or the leguminB4 promoter (LeB4; Baeumlein et al., 1992, Plant Journal, 2 (2):233-9)as well as promoters conferring seed specific expression in monocotplants like maize, barley, wheat, rye, rice etc. Suitable promoters tonote are the lpt2 or lpt1-gene promoter from barley (WO 95/15389 and WO95/23230) or those described in WO 99/16890 (promoters from the barleyhordein-gene, the rice glutelin gene, the rice oryzin gene, the riceprolamin gene, the wheat gliadin gene, wheat glutelin gene, the maizezein gene, the oat glutelin gene, the Sorghum kasirin-gene, the ryesecalin gene).

Also especially suited are promoters that confer plastid-specific geneexpression as plastids are the compartment where precursors and some endproducts of lipid biosynthesis are synthesized. Suitable promoters suchas the viral RNA-polymerase promoter are described in WO 95/16783 and WO97/06250 and the clpP-promoter from Arabidopsis described in WO99/46394.

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to PSE mRNA. Regulatory sequences operatively linkedto a nucleic acid cloned in the antisense orientation can be chosenwhich direct the continuous expression of the antisense RNA molecule ina variety of cell types, for instance viral promoters and/or enhancers,or regulatory sequences can be chosen which direct constitutive, tissuespecific or cell type specific expression of antisense RNA. Theantisense expression vector can be in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense nucleic acids areproduced under the control of a high efficiency regulatory region, theactivity of which can be determined by the cell type into which thevector is introduced. For a discussion of the regulation of geneexpression using antisense genes see Weintraub, H. et al., Antisense RNAas a molecular tool for genetic analysis, Reviews—Trends in Genetics,Vol. 1(1) 1986 and Mol et al., 1990, FEBS Letters 268:427-430.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, anASE can be expressed in bacterial cells such as C. glutamicum, insectcells, fungal cells or mammalian cells (such as Chinese hamster ovarycells (CHO) or COS cells), algae, ciliates, plant cells, fungi or othermicroorganisms like C. glutamicum. Other suitable host cells are knownto those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection”, conjugation andtransduction are intended to refer to a variety of art-recognizedtechniques for introducing foreign nucleic acid (e.g., DNA) into a hostcell, including calcium phosphate or calcium chloride coprecipitation,DEAE-dextran-mediated transfection, lipofection, natural competence,chemical-mediated transfer, or electroporation. Suitable methods fortransforming or transfecting host cells including plant cells can befound in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nded., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1989) and other laboratory manuals such asMethods in Molecular Biology, 1995, Vol. 44, Agrobacterium protocols,ed: Gartland and Davey, Humana Press, Totowa, N.J.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as hygromycin and methotrexate or, in plants, those whichconfer resistance towards a herbicide such as imidazolinones,sulfonylurea, glyphosate or glufosinate. Nucleic acid encoding aselectable marker can be introduced into a host cell on the same vectoras that encoding an ASE or can be introduced on a separate vector. Cellsstably transfected with the introduced nucleic acid can be identifiedby, for example, drug selection (e.g., cells that have incorporated theselectable marker gene will survive, while the other cells die).

To create a homologously-recombinant microorganism, a vector is preparedwhich contains at least a portion of an ASE gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the ASE gene. Preferably, this ASE gene is anIsochrysis galbana ASE gene, but it can be a homolog from a relatedplant or even from an alga, mammalian, yeast, or insect source. In apreferred embodiment, the vector is designed such that, upon homologousrecombination, the endogenous ASE gene is functionally disrupted (i.e.,no longer encodes a functional protein; also referred to as a knock-outvector). Alternatively, the vector can be designed such that, uponhomologous recombination, the endogenous ASE gene is mutated orotherwise altered but still encodes a functional protein (e.g., theupstream regulatory region can be altered to thereby alter theexpression of the endogenous ASE). To create a point mutation viahomologous recombination also DNA-RNA hybrids can be used known aschimeraplasty known from Cole-Strauss et al. 1999, Nucleic AcidsResearch 27(5):1323-1330 and Kmiec, Gene therapy. 1999, AmericanScientist. 87(3):240-247.

In the homologously-recombinant vector, the altered portion of the ASEgene is flanked at its 5′ and 3′ ends by additional nucleic acid of theASE gene to allow for homologous recombination to occur between theexogenous ASE gene carried by a vector and an endogenous ASE gene in amicroorganism or plant. The additional flanking ASE nucleic acid is ofsufficient length for successful homologous recombination with theendogenous gene. Typically, several hundreds of basepairs up tokilobases of flanking DNA (both at the 5′ and 3′ ends) are included inthe vector (see e.g., Thomas, K. R., and Capecchi, M. R. (1987) Cell 51:503 for a description of homologous recombination vectors or Strepp etal., 1998, PNAS, 95 (8):4368-4373 for cDNA based recombination inIsochrysis galbana). The vector is introduced into a microorganism orplant cell (e.g., via Agrobacterium mediated gene transfer, biolistictransformation, polyethylene glycol or other applicable methods) andcells in which the introduced ASE gene has homologously recombined withthe endogenous ASE gene are selected, using art-known techniques. In thecase of plant cells the AHAS gene described in Ott et al., J. Mol. Biol.1996, 263:359-360 is especially suitable for marker gene expression andresistance towards imidazolinone or sulfonylurea type herbicides.

In another embodiment, recombinant organisms such as microorganisms canbe produced which contain selected systems which allow for regulatedexpression of the introduced gene. For example, inclusion of an ASE genein a vector placing it under control of the Lac operon permitsexpression of the ASE gene only in the presence of IPTG. Such regulatorysystems are well known in the art. Recombinant organisms means anorganism which comprises an inventive nucleic acid sequence, a geneconstruct or a vector in the cell or inside the genome at a place whichis not the “natural” place or at the “natural” place but modified in amanner which is not the natural manner; that means the coding sequenceis modified and/or the regulatory sequence is modified. Modified meanssingle nucleotides or one or more codons are changed in comparison tothe natural sequence, preferably one ore more codons, more preferablyone to six codons.

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) an ASE. Analternate method can be applied in addition in plants by the directtransfer of DNA into developing flowers via electroporation orAgrobacterium-mediated gene transfer. Accordingly, the invention furtherprovides methods for producing ASEs using the host cells of theinvention. In one embodiment, the method comprises culturing the hostcell of the invention (into which a recombinant expression vectorencoding an ASE has been introduced, or into whose genome has beenintroduced a gene encoding a wild-type or altered ASE) in a suitablemedium until ASE is produced. In another embodiment, the method furthercomprises isolating ASEs from the medium or the host cell.

Host cells suitable in principle to take up the nucleic acid of theinvention, the novel gene construct or the inventive vector are allprokaryotic or eukaryotic organisms. The host organisms advantageouslyused are organisms such as bacteria, fungi, yeasts, animal or plantcells. Additional advantageous organisms are animals or preferablyplants or parts thereof. Fungi, yeasts or plants are preferably used,particularly preferably fungi or plants, very particularly preferablyplants such as oilseed plants containing high amounts of lipid compoundssuch as rapeseed, evening primrose, canola, peanut, linseed, soybean,safflower, sunflower, borage or plants such as maize, wheat, rye, oat,triticale, rice, barley, cotton, manihot, pepper, tagetes, solanaceousplants such as potato, tobacco, eggplant, and tomato, Vicia species,pea, alfalfa, bushy plants (coffee, cacao, tea), Salix species, trees(oil palm, coconut) and perennial grasses and forage crops. Particularlypreferred plants of the invention are oilseed plants such as soybean,peanut, rapeseed, canola, sunflower, safflower, trees (oil palm,coconut).

D. Isolated ASE

Another aspect of the invention pertains to isolated ASEs, andbiologically active portions thereof. An “isolated” or “purified”protein or biologically active portion thereof is substantially free ofcellular material when produced by recombinant DNA techniques, orchemical precursors or other chemicals when chemically synthesized. Thelanguage “substantially free of cellular material” includes preparationsof ASE in which the protein is separated from cellular components of thecells in which it is naturally or recombinantly produced. In oneembodiment, the language “substantially free of cellular material”includes preparations of ASE having less than about 30% (by dry weight)of non-ASE (also referred to herein as a “contaminating protein”), morepreferably less than about 20% of non-ASE, still more preferably lessthan about 10% of non-ASE, and most preferably less than about 5% ofnon-ASE. When the ASE or biologically active portion thereof isrecombinantly produced, it is also substantially free of culture medium,i.e., culture medium represents less than 20%, more preferably less than10%, and most preferably less than about 5% of the volume of the proteinpreparation. The language “substantially free of chemical precursors orother chemicals' includes preparations of ASE in which the protein isseparated from chemical precursors or other chemicals which are involvedin the synthesis of the protein. In one embodiment, the language“substantially free of chemical precursors or other chemicals” includespreparations of ASE having less than about 30% (by dry weight) ofchemical precursors or non-ASE chemicals, more preferably less thanabout 20% chemical precursors or non-ASE chemicals, still morepreferably less than about 10% chemical precursors or non-ASE chemicals,and most preferably less than about 5% chemical precursors or non-ASEchemicals. In preferred embodiments, isolated proteins or biologicallyactive portions thereof lack contaminating proteins from the sameorganism from which the ASE is derived. Typically, such proteins areproduced by recombinant expression of, for example, an Isochrysisgalbana ASE in other plants than Isochrysis galbana or microorganismssuch as C. glutamicum or ciliates, algae or fungi.

An isolated ASE of the invention or a portion thereof can participate inthe metabolism of compounds involved in the construction of cellularmembranes in Isochrysis galbana, or in the transport of molecules acrossthese membranes, or has one or more of the activities set forth in Tab.2. In preferred embodiments, the protein or portion thereof comprises anamino acid sequence which is sufficiently homologous to an amino acidsequence of SEQ ID NO: 2 such that the protein or portion thereofmaintains the ability to participate in the metabolism of compoundsnecessary for the construction of cellular membranes in Isochrysisgalbana, or in the transport of molecules across these membranes. Theportion of the protein is preferably a biologically active portion asdescribed herein. In another preferred embodiment, an ASE of theinvention has an amino acid sequence shown in SEQ ID NO: 2. In anotherpreferred embodiment, the ASE has an amino acid sequence which isencoded by a nucleotide sequence which hybridizes, e.g., hybridizesunder stringent conditions, to a nucleotide sequence of SEQ ID NO: 1. Inanother preferred embodiment, the ASE has an amino acid sequence whichis encoded by a nucleotide sequence that is at least about 50-60%,preferably at least about 60-70%, more preferably at least about 70-80%,80-90%, 90-95%, and even more preferably at least about 96%, 97%, 98%,99% or more homologous to one of the amino acid sequences of SEQ ID NO:2. The preferred ASEs of the present invention also preferably possessat least one of the ASE activities described herein. For example, apreferred ASE of the present invention includes an amino acid sequenceencoded by a nucleotide sequence which hybridizes, e.g., hybridizesunder stringent conditions, to a nucleotide sequence of SEQ ID NO: 1,and which can participate in the metabolism of compounds necessary forthe construction of cellular membranes in Isochrysis galbana, or in thetransport of molecules across these membranes, or which has one or moreof the activities set forth in Tab. 2.

In other embodiments, the ASE is substantially homologous to an aminoacid sequence of SEQ ID NO: 2 and retains the functional activity of theprotein of one of the sequences of SEQ ID NO: 2 yet differs in aminoacid sequence due to natural variation or mutagenesis, as described indetail in subsection I above. Accordingly, in another embodiment, theASE is a protein which comprises an amino acid sequence which is atleast about 50-60%, preferably at least about 60-70%, and morepreferably at least about 70-80, 80-90, 90-95%, and most preferably atleast about 96%, 97%, 98%, 99% or more homologous to an entire aminoacid sequence of SEQUENCE ID NO: 2 and which has at least one of the ASEactivities described herein. In another embodiment, the inventionpertains to a full Isochrysis galbana protein which is substantiallyhomologous to an entire amino acid sequence of SEQ ID NO: 2.

Biologically active portions of an ASE include peptides comprising aminoacid sequences derived from the amino acid sequence of an ASE, e.g., theamino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence ofa protein homologous to an ASE, which include fewer amino acids than afull length ASE or the full length protein which is homologous to anASE, and exhibit at least one activity of an ASE. Typically,biologically active portions (peptides, e.g., peptides which are, forexample, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or moreamino acids in length) comprise a domain or motif with at least oneactivity of an ASE. Moreover, other biologically active portions, inwhich other regions of the protein are deleted, can be prepared byrecombinant techniques and evaluated for one or more of the activitiesdescribed herein. Preferably, the biologically active portions of an ASEinclude one or more selected domains/motifs or portions thereof havingbiological activity.

ASEs are preferably produced by recombinant DNA techniques. For example,a nucleic acid molecule encoding the protein is cloned into anexpression vector (as described above), the expression vector isintroduced into a host cell (as described above) and the ASE isexpressed in the host cell. The ASE can then be isolated from the cellsby an appropriate purification scheme using standard proteinpurification techniques. Alternatively to recombinant expression, anASE, polypeptide, or peptide can be synthesized chemically usingstandard peptide synthesis techniques. Moreover, native ASE can beisolated from cells (e.g., endothelial cells), for example using ananti-ASE antibody, which can be produced by standard techniquesutilizing an ASE of this invention or fragment thereof.

The invention also provides ASE chimeric or fusion proteins. As usedherein, an ASE “chimeric protein” or “fusion protein” comprises an ASEpolypeptide operatively linked to a non-ASE polypeptide. An “ASEpolypeptide” refers to a polypeptide having an amino acid sequencecorresponding to an ASE, whereas a “non-ASE polypeptide” refers to apolypeptide having an amino acid sequence corresponding to a proteinwhich is not substantially homologous to the ASE, e.g., a protein whichis different from the ASE and which is derived from the same or adifferent organism. Within the fusion protein, the term “operativelylinked” is intended to indicate that the ASE polypeptide and the non-ASEpolypeptide are fused to each other so that both sequences fulfill thepredicted function ascribed to the sequence used. The non-ASEpolypeptide can be fused to the N-terminus or C-terminus of the ASEpolypeptide. For example, in one embodiment the fusion protein is aGST-ASE fusion protein in which the ASE sequences are fused to theC-terminus of the GST sequences. Such fusion proteins can facilitate thepurification of recombinant ASEs. In another embodiment, the fusionprotein is an ASE containing a heterologous signal sequence at itsN-terminus. In certain host cells (e.g., mammalian host cells),expression and/or secretion of an ASE can be increased through use of aheterologous signal sequence.

An ASE chimeric or fusion protein of the invention is produced bystandard recombinant DNA techniques. For example, DNA fragments codingfor the different polypeptide sequences are ligated together in-frame inaccordance with conventional techniques, for example by employingblunt-ended or sticky-ended termini for ligation, restriction enzymecleavage to provide for appropriate termini, filling-in of cohesive endsas appropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. In another embodiment, the fusion genecan be synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,for example, Current Protocols in Molecular Biology, eds. Ausubel et al.John Wiley & Sons: 1992). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide). An ASE-encoding nucleic acid can be cloned into such anexpression vector such that the fusion moiety is linked in-frame to theASE.

Homologs of the ASE can be generated by mutagenesis, e.g., discretepoint mutation or truncation of the ASE. As used herein, the term“homolog” refers to a variant form of the ASE which acts as an agonistor antagonist of the activity of the ASE. An agonist of the ASE canretain substantially the same, or a subset, of the biological activitiesof the ASE. An antagonist of the ASE can inhibit one or more of theactivities of the naturally occurring form of the ASE, by, for example,competitively binding to a downstream or upstream member of the cellmembrane component metabolic cascade which includes the ASE, or bybinding to an ASE which mediates transport of compounds across suchmembranes, thereby preventing translocation from taking place.

In an alternative embodiment, homologs of the ASE can be identified byscreening combinatorial libraries of mutants, e.g., truncation mutants,of the ASE for ASE agonist or antagonist activity. In one embodiment, avariegated library of ASE variants is generated by combinatorialmutagenesis at the nucleic acid level and is encoded by a variegatedgene library. A variegated library of ASE variants can be produced by,for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential ASE sequences is expressible as individual polypeptides, or,alternatively, as a set of larger fusion proteins (e.g., for phagedisplay) containing the set of ASE sequences therein. There are avariety of methods which can be used to produce libraries of potentialASE homologs from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be performed in an automaticDNA synthesizer, and the synthetic gene then ligated into an appropriateexpression vector. Use of a degenerate set of genes allows for theprovision, in one mixture, of all of the sequences encoding the desiredset of potential ASE sequences. Methods for synthesizing degenerateoligonucleotides are known in the art (see, e.g., Narang, S. A. (1983)Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323;Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic AcidRes. 11:477.

In addition, libraries of fragments of the ASE can be used to generate avariegated population of ASE fragments for screening and subsequentselection of homologs of an ASE. In one embodiment, a library of codingsequence fragments can be generated by treating a double stranded PCRfragment of an ASE coding sequence with a nuclease under conditionswherein nicking occurs only about once per molecule, denaturing thedouble stranded DNA, renaturing the DNA to form double stranded DNAwhich can include sense/antisense pairs from different nicked products,removing single stranded portions from reformed duplexes by treatmentwith S1 nuclease, and ligating the resulting fragment library into anexpression vector. By this method, an expression library can be derivedwhich encodes N-terminal, C-terminal and internal fragments of varioussizes of the ASE.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of ASE homologs. The mostwidely used techniques, which are amenable to high-throughput analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of the desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a newtechnique which enhances the frequency of functional mutants in thelibraries, can be used in combination with the screening assays toidentify ASE homologs (Arkin and Yourvan (1992) PNAS 89:7811-7815;Delgrave et al. (1993) Protein Engineering 6(3):327-331).

In another embodiment, cell based assays can be exploited to analyze avariegated ASE library, using further methods well known in the art.

E. Uses and Methods of the Invention

The nucleic acid molecules, proteins, protein homologs, fusion proteins,primers, vectors, and host cells described herein can be used in one ormore of the following methods: identification of Isochrysis galbana andrelated organisms; mapping of genomes of organisms related to Isochrysisgalbana; identification and localization of Isochrysis galbana sequencesof interest; evolutionary studies; determination of ASE regions requiredfor function; modulation of an ASE activity; modulation of themetabolism of one or more cell membrane components; modulation of thetransmembrane transport of one or more compounds; and modulation ofcellular production of a desired compound, such as a fine chemical,including PUFAs.

The ASE nucleic acid molecules of the invention have a variety of uses.First, they may be used to identify an organism as being Isochrysisgalbana or a close relative thereof. Also, they may be used to identifythe presence of Isochrysis galbana or a relative thereof in a mixedpopulation of microorganisms. The invention provides the nucleic acidsequences of a number of Isochrysis galbana genes; by probing theextracted genomic DNA of a culture of a unique or mixed population ofmicroorganisms under stringent conditions with a probe spanning a regionof an Isochrysis galbana gene which is unique to this organism, one canascertain whether this organism is present. Although Isochrysis galbanaitself is not used for the commercial production of polyunsaturatedacids, algae are the only known plants beside mosses that produce morethen a few percent of their total lipids as PUFAs. Therefore DNAsequences related to ASEs are especially suited to be used for PUFAproduction in other organisms.

Further, the nucleic acid molecules and protein molecules of theinvention may serve as markers for specific regions of the genome. Thishas utility not only in the mapping of the genome, but also forfunctional studies of Isochrysis galbana proteins. For example, toidentify the region of the genome to which a particular Isochrysisgalbana DNA-binding protein binds, the Isochrysis galbana genome couldbe digested, and the fragments incubated with the DNA-binding protein.Those which bind the protein may be additionally probed with the nucleicacid molecules of the invention, preferably with readily detectablelabels; binding of such a nucleic acid molecule to the genome fragmentenables the localization of the fragment on the genome map of Isochrysisgalbana, and, when performed multiple times with different enzymes,facilitates a rapid determination of the nucleic acid sequence to whichthe protein binds. Further, the nucleic acid molecules of the inventionmay be sufficiently homologous to the sequences of related species suchthat these nucleic acid molecules may serve as markers for theconstruction of a genomic map for related algae, such as Isochrysisgalbana.

The ASE nucleic acid molecules of the invention are also useful forevolutionary and protein structural studies. The metabolic and transportprocesses in which the molecules of the invention participate areutilized by a wide variety of prokaryotic and eukaryotic cells; bycomparing the sequences of the nucleic acid molecules of the presentinvention to those encoding similar enzymes from other organisms, theevolutionary relatedness of the organisms can be assessed. Similarly,such a comparison permits an assessment of which regions of the sequenceare conserved and which are not, which may aid in determining thoseregions of the protein which are essential for the functioning of theenzyme. This type of determination is of value for protein engineeringstudies and may give an indication of what the protein can tolerate interms of mutagenesis without losing function.

Manipulation of the ASE nucleic acid molecules of the invention mayresult in the production of ASEs having functional differences from thewild-type ASEs. These proteins may be improved in efficiency oractivity, may be present in greater numbers in the cell than is usual,or may be decreased in efficiency or activity. Improved efficiency oractivity means for example the enzyme has a higher selectivity and/oractivity than the original enzyme, preferably at least 10% higher,particularly preferably at least 20% higher activity, most particularlypreferably at least 30% higher activity.

There are a number of mechanisms by which the alteration of an ASE ofthe invention may directly affect the yield, production, and/orefficiency of production of a fine chemical incorporating such analtered protein. Recovery of fine chemical compounds from large-scalecultures of ciliates, algae or fungi is significantly improved if thecell secretes the desired compounds, since such compounds may be readilyisolated from the culture medium (as opposed to extracted from the massof cultured cells). Otherwise purification can be improved if preferablythe cell stores compounds in a specialized compartment having a kind ofconcentrating mechanism in vivo. In the case of plants expressing ASEsincreased transport can lead to improved partitioning within the planttissue and organs. By either increasing the number or the activity oftransporter molecules which export fine chemicals from the cell, it maybe possible to increase the amount of the produced fine chemical whichis present in the extracellular medium, thus permitting greater ease ofharvesting and purification or, in the case of plants, more efficientpartitioning. Conversely, in order to efficiently overproduce one ormore fine chemicals, increased amounts of the cofactors, precursormolecules, and intermediate compounds for the appropriate biosyntheticpathways are required. By increasing the number and/or activity oftransporter proteins involved in the import of nutrients, such as carbonsources (i.e., sugars), nitrogen sources (i.e., amino acids, ammoniumsalts), phosphate, and sulfur, it may be possible to improve theproduction of a fine chemical, due to the removal of any nutrient supplylimitations on the biosynthetic process. Fatty acids such as PUFAs andlipids containing PUFAs are themselves desirable fine chemicals, so byoptimizing the activity or increasing the number of one or more ASEs ofthe invention which participate in the biosynthesis of these compounds,or by impairing the activity of one or more genes which are involved inthe degradation of these compounds, it may be possible to increase theyield, production, and/or efficiency of production of fatty acid andlipid molecules in ciliates, algae, plants, fungi, yeasts or othermicroorganisms.

The engineering of one or more ASE genes of the invention may alsoresult in ASEs having altered activities which indirectly impact theproduction of one or more desired fine chemicals from algae, plants,ciliates or fungi. For example, the normal chemical processes ofmetabolism result in the production of a variety of waste products(e.g., hydrogen peroxide and other reactive oxygen species) which mayactively interfere with these same metabolic processes (for example,peroxynitrite is known to nitrate tyrosine side chains, therebyinactivating some enzymes having tyrosine in the active site (Groves, J.T. (1999) Curr. Opin. Chem. Biol. 3(2): 226-235). While these wasteproducts are typically excreted, cells utilized for large-scalefermentative production are optimized for the overproduction of one ormore fine chemicals, and thus may produce more waste products than istypical for a wild-type cell. By optimizing the activity of one or moreASEs of the invention, it may be possible to improve the viability ofthe cell and to maintain efficient metabolic activity, thereby improvingthe production of the desired product such as PUFAs. Also, the presenceof high intracellular levels of the desired fine chemical may actuallybe toxic to the cell, so by increasing the ability of the cell tosecrete these compounds, one may further improve the viability of thecell.

Further, the ASEs of the invention may be manipulated such that therelative amounts of various lipid and fatty acid molecules are altered.This may have a profound effect on the lipid composition of the membraneof the cell. Since each type of lipid has different physical properties,an alteration in the lipid composition of a membrane may significantlyalter membrane fluidity. Changes in membrane fluidity can impact thetransport of molecules across the membrane, which, as previouslyexplicated, may modify the export of waste products or the produced finechemical or the import of necessary nutrients. Such membrane fluiditychanges may also profoundly affect the integrity of the cell; cells withrelatively weaker membranes are more susceptible to abiotic and bioticstress conditions which may damage or kill the cell. By manipulatingASEs involved in the production of fatty acids and lipids for membraneconstruction such that the resulting membrane has a membrane compositionmore amenable to the environmental conditions extant in the culturesutilized to produce fine chemicals, a greater proportion of the cellsshould survive and multiply. Greater numbers of producing cells shouldtranslate into greater yields, production, or efficiency of productionof the fine chemical from the culture.

The aforementioned mutagenesis strategies for ASEs to result inincreased yields of a fine chemical are not meant to be limiting;variations of these strategies will be readily apparent to one skilledin the art. Using such strategies, and incorporating the mechanismsdisclosed herein, the nucleic acid molecules and protein molecules ofthe invention may be utilized to generate algae, ciliates, plants,animals, fungi or other microorganisms like C. glutamicum expressingmutated ASE nucleic acid and protein molecules such that the yield,production, and/or efficiency of production of a desired compound isimproved. This desired compound may be any natural product of algae,ciliates, plants, animals or fungi, which includes the final products ofbiosynthesis pathways and intermediates of naturally-occurring metabolicpathways, as well as molecules which do not naturally occur in themetabolism of said cells, but which are produced by the cells of theinvention.

Another embodiment of the invention is a method for production of PUFAs,said method comprising growing an organism which comprises a nucleicacid of the invention, a gene construct of the invention or a vector ofthe invention which encodes a polypeptide which elongates α-linolenicacid (C_(18:3 d9, 12, 15)) by at least two carbon atoms but notγ-linolenic acid (C_(18:3 d6, 9, 12)), under conditions whereby PUFAsare produced in said organism. Preferably the method comprises thegrowing of an organism which comprises a nucleotide sequence whichencodes a polypeptide which elongates α-linolenic acid(C_(18:3 d9, 12, 15)) by at least two carbon atoms whereas γ-linolenicacid (C_(18:3 d6, 9, 12)) is not elongated, selected from the groupconsisting of

-   a) a nucleic acid sequence depicted in SEQ ID NO: 1,-   b) a nucleic acid sequence which encodes a polypeptide depicted in    SEQ ID NO: 2,-   c) derivatives of the sequence depicted in SEQ ID NO: 1, which    encodes polypeptides having at least 50% homology to the sequence    encoding amino acid sequences depicted in SEQ ID NO: 2 and which    sequences function as an elongase.

More preferably the nucleic acid sequence is derived from a plant,preferably from the genus Isochrysis. The used sequence codes for apolypeptide which elongates Δ9 fatty acids.

The PUFAs produced by this method are preferably C₂₀ or C₂₂ fatty acidmolecules having at least two double bonds in the fatty acid molecule,preferably at least three double bonds.

Organisms which are useful in the inventive method for the production ofPUFAs are microorganism such as bacteria like Gram-positive orG@@@@ram-negative bacteria or preferably blue algae, ciliates such asColpidium or Stylonichia, fungi such as Mortierella or Thraustochytriumor Schizochytrium, algae such as Phaeodactylum, and/or plants likemaize, wheat, rye, oats, triticale, rice, barley, soybean, peanut,cotton, Brassica species like rapeseed, canola and turnip rape, linseed,pepper, sunflower, borage, evening primrose and tagetes, solanaceousplants like potato, tobacco, eggplant, and tomato, Vicia species, pea,manihot, alfalfa, bushy plants (coffee, cacao, tea), Salix species,trees (oil palm, coconut) and perennial grasses and forage crops, eitherdirectly, e.g., mosses or other plants where overexpression oroptimization of a fatty acid biosynthesis protein has a direct impact onthe yield, production, and/or efficiency of production of the fatty acidfrom modified organisms.

PUFAs can be produced in the inventive process in the form of an oil,lipid or free fatty acid. PUFAs produced by this method can be isolatedby harvesting the organisms either from the culture in which they weregrowing or from the field, disrupting and/or extracting the harvestedmaterial with an organic solvent. From said solvent the oil containinglipids, phospholipids, sphingolipids, glycolipids, triacylglycerolsand/or free fatty acids with a higher content of PUFAs can be isolated.By basic or acid hydrolysis of the lipids, phospholipids, sphingolipids,glycolipids or triacylglycerols, the free fatty acids with a highercontent of PUFAs can be isolated. Higher content of PUFAs means at least1%, preferably 10%, particularly preferably 20%, most particularlypreferably 40% more PUFAs than the original organism which has noadditional nucleic acid coding for the inventive elongase.

Besides the abovementioned methods, plant lipids are extractedpreferably from plant material as described by Cahoon et al. (1999) PNAS96 (22): 12935-12940 and Browse et al. (1986) Analytic Biochemistry 152:141-145. Qualitative and quantitative lipid or fatty acid analysis isdescribed in Christie, William W., Advances in Lipid Methodology,Ayr/Scotland: Oily Press. —(Oily Press Lipid Library; 2); Christie,William W., Gas Chromatography and Lipids. A Practical Guide—Ayr,Scotland: Oily Press, 1989 Repr. 1992—IX, 307 p. —(Oily Press LipidLibrary; 1); “Progress in Lipid Research, Oxford: Pergamon Press,1(1952)-16(1977) under the title: Progress in the Chemistry of Fats andOther Lipids.

PUFAs produced by this method are preferably C₂₀ or C₂₂ fatty acidmolecules having at least two double bonds in the fatty acid molecule,preferably three to four double bonds, particularly preferably threedouble bonds. Such C₂₀ or C₂₂ fatty acid molecules can be isolated fromthe organism in the form of an oil, lipid or free fatty acid. Organismswhich are useful are for example the ones mentioned above. Preferredorganisms are transgenic plants.

One embodiment of the invention is oils, lipids or fatty acids orfractions thereof produced by the method described above, particularlypreferably an oil, lipid or fatty acid composition comprising PUFAsderived from transgenic plants.

A further embodiment of the invention is the use of said oil, lipid orfatty acid composition in feed, food, cosmetics or pharmaceuticals.

An additional embodiment of the invention is a monoclonal or polyclonalantibody which specifically interacts with a polypeptide encoded by theinventive nucleic acid sequence described above and which is produced bya method known by the skilled worker.

A further embodiment of the invention is a kit comprising an inventivenucleotide sequence, a gene construct as claimed, a vector as claimed oran antibody as described above. Said kit is useful for example for theidentification of the protein, the nucleic acid sequence.

The aforementioned mutagenesis strategies for ASEs to result inincreased yields of a fine chemical are not meant to be limiting;variations on these strategies will be readily apparent to one skilledin the art. Using such strategies, and incorporating the mechanismsdisclosed herein, the nucleic acid molecules and protein molecules ofthe invention may be utilized to generate algae, ciliates, plants, fungior other microorganisms like C. glutamicum expressing mutated ASEnucleic acid and protein molecules such that the yield, production,and/or efficiency of production of a desired compound is improved. Thisdesired compound may be any natural product of algae, ciliates, plants,fungi or C. glutamicum, which includes the final products ofbiosynthesis pathways and intermediates of naturally-occurring metabolicpathways, as well as molecules which do not naturally occur in themetabolism of said cells, but which are produced by said cells of theinvention.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patent applications, patents, and published patent applications citedthroughout this application are hereby incorporated by reference.

EXAMPLES Example 1 General Processes

a) Cloning Processes and General Methods

Cloning processes such as, for example, restriction cleavages, agarosegel electrophoresis, purification of DNA fragments, transfer of nucleicacids to nitrocellulose and nylon membranes, linkage of DNA fragments,transformation of Escherichia coli and yeast cells, growth of bacteriaand sequence analysis of recombinant DNA were carried out as describedin Sambrook et al. (1989) (Cold Spring Harbor Laboratory Press: ISBN0-87969-309-6) or Kaiser, Michaelis and Mitchell (1994) “Methods inYeast Genetics” (Cold Spring Harbor Laboratory Press: ISBN0-87969-451-3). Transformation and cultivation of algae such asChlorella or Phaeodactylum are performed as described by El-Sheekh(1999), Biologia Plantarum 42: 209-216; Apt et al. (1996), Molecular andGeneral Genetics 252 (5): 872-9.

b) Chemicals:

The chemicals used were obtained, if not mentioned otherwise in thetext, in p.a. quality from the companies Fluka (Neu-Ulm), Merck(Darmstadt), Roth (Karlsruhe), Serva (Heidelberg) and Sigma(Deisenhofen). Solutions were prepared using purified, pyrogen-freewater, designated as H₂O in the following text, from a Milli-Q watersystem water purification plant (Millipore, Eschborn). Restrictionendonucleases, DNA-modifying enzymes and molecular biology kits wereobtained from the companies AGS (Heidelberg), Amersham (Braunschweig),Biometra (Göttingen), Boehringer (Mannheim), Genomed (Bad Oeynhausen),New England Biolabs (Schwalbach/Taunus), Novagen (Madison, Wis., USA),Perkin-Elmer (Weiterstadt), Pharmacia (Freiburg), Qiagen (Hilden) andStratagene (Amsterdam, Netherlands). They were used, if not mentionedotherwise, according to the manufacturer's instructions.

c) Algal Material

For this study, algae of the species Isochrysis galbana CCAP 927/1 wereused, obtained from the Culture Collection of Algae and Protozoa, Centrefor Coastal and Marine Sciences, Dunstaffnage Marine Laboratory, Oban,Argyll; UK.

Cultivation of Algae

Isochrysis galbana was cultured using the f/2 medium containing 10%organic medium as described by Guillard, R. R. L. [1975; Culture ofphytoplankton for feeding marine invertebrates. In: Smith, W. L. andChanley, M. H. (Eds.) Culture of marine Invertebrate animals, NY PlenumPress, pp. 29-60.]. Isochrysis galbana was cultured at 14° C. undercontinuous light and a light intensity of 30 microEinstein in glassvessels with shaking at 100 rpm.

The f/2 medium consists of:

995.5 ml artificial sea water containing:

-   -   1 ml NaNO₃ (75 g/l),    -   1 ml NaH₂PO₄ (5 g/l),    -   1 ml trace element solution,    -   1 ml Tris/Cl pH 8.0,    -   0.5 ml f/2 vitamin solution    -   Trace element solution:        -   Na₂EDTA (4.36 g/l),        -   FeCl₃ (3.15 g/l),    -   Primary trace elements:        -   CuSO₄ (10 g/l),        -   ZnSO₄ (22 g/l),        -   CoCl₂ (10 g/l),        -   MnCl₂ (18 g/l),        -   NaMoO₄ (6.3 g/l)    -   f/2 vitamin solution:        -   biotin: 10 mg/l,        -   thiamine 200 mg/l,        -   vit B12 0.1 mg/l

Org. Medium:

-   -   Na acetate (1 g/l),    -   glucose (6 g/l),    -   Na succinate (3 g/l),    -   Bacto-Tryptone (4 g/l),    -   Yeast extract (2 g/l)

Example 2 DNA Isolation from Algae

The details for the isolation of total DNA relate to the working up ofone gram fresh weight of material harvested by filtration.

CTAB Buffer:

-   -   2% (w/v) N-cetyl-N,N,N-trimethylammonium bromide (CTAB);    -   100 mM Tris HCl pH 8.0;    -   1.4 M NaCl;    -   20 mM EDTA.        N-Laurylsarcosine Buffer:    -   10% (w/v) N-laurylsarcosine;    -   100 mM Tris HCl pH 8.0;    -   20 mM EDTA.

The material was homogenized under liquid nitrogen with quartz sand in amortar to give a fine powder and transferred to 2 ml Eppendorf cups. Thefrozen material was then covered with a layer of 1 ml of decompositionbuffer (1 ml CTAB buffer, 100 ml of N-laurylsarcosine buffer, 20 ml ofbeta-mercaptoethanol and 10 ml of proteinase K solution, 10 mg/ml) andincubated at 60° C. for one hour with continuous shaking. The homogenateobtained was distributed into two Eppendorf vessels (2 ml) and extractedtwice by shaking with the same volume of chloroform/isoamyl alcohol(24:1). For phase separation, centrifugation was carried out at 8000×gand room temperature for 15 min in each case. The DNA was thenprecipitated at 70° C. for 30 min. The precipitated DNA was sedimentedat 4° C. and 10,000 g for 30 min and resuspended in 100 microliters ofTE buffer (Sambrook et al., 1989, Cold Spring Harbor Laboratory Press:ISBN 0-87969-309-6). For further purification, the DNA was treated withNaCl (1.2 M final concentration) and precipitated again at 70° C. for 30min using twice the volume of absolute ethanol. After a washing stepwith 70% ethanol, the DNA was dried and subsequently taken up in 50microliters of H₂O+DNase free RNase (50 mg/ml final concentration). TheDNA was dissolved overnight at 4° C. and the RNase digestion wassubsequently carried out at 37° C. for 1 h. Storage of the DNA tookplace at 4° C.

Example 3 Isolation of Total RNA and Poly(A)+RNA from Algae

For the investigation of transcripts, both total RNA and poly(A)+RNAwere isolated.

Algal cultures were harvested by centrifugation at 3000 g for 5 minutes.The pellets were immediately frozen in liquid nitrogen (−70° C.). Algalmaterial (1 g) was homogenized with a pestle in a mortar under liquidnitrogen. The material was desintegrated to homogeneity in two volumesof buffer which was TriPure™ Isolation Reagent (Roche). The total RNAwas then isolated following the manufacturer's protocol.

Isolation of Poly(A)+RNA was carried out using Amersham Pharmacia mRNAIsolation Kit following the instructions of the manufacturer's protocol.

After determination of the concentration of the RNA or of thepoly(A)+RNA, the RNA was precipitated by addition of 1/10 volumes of 3 Msodium acetate pH 4.6 and 2 volumes of ethanol and stored at −70° C.

Example 4 cDNA Library Construction

Double stranded cDNA was synthesised using the cDNA Synthesis Kit fromStratagene following the manufacturer's protocol. It was then passedthrough a Sephacyl S-400 Spun Column from a cDNA Synthesis Kit (AmershamPharmacia) to remove adapters and smaller molecules. cDNA eluted fromthe column was phenol extracted, ethanol precipitated and ligated to thearms of the Uni-Zap vector and packed into λ phages using Ready-To-GoLambda Packaging Kit (Amersham Pharmacia Biotech) following themanufacturer's instructions. A library of 1×10⁶ pfu was obtained withthe majority of the inserts ranging from 0.4-2 kb.

Example 5 Identification of the ASE1 Gene and Analysis of the cDNA-cloneIg_ASE1

From an alignment of known elongase sequences (from M. alpina, S.cerevisiae (Elo1, Elo2, Elo3), C. elegans (F56H11.4, F41H10.8)) thecommon motif MYXYYFL (SEQ ID NO:3) was chosen for oligonucleotidedesign.

The reverse complement oligo

5′-AAAAAATAATAIIIGTACAT-3′ (SEQ ID NO:4)

5′-AGGAAGTAGTAIIIATACAT-3′ (SEQ ID NO:5) (I=deoxyinosine)

was synthesised and used in touchdown PCR with a universal T3 promoterprimer (5′-AATTAACCCTCACTAAAGGG-3′) (SEQ ID NO:6) using an Isochrysisgalbana cDNA library as template.

The PCR conditions were:

94° C. for 3 min (1 cycle)

94° C. for 15 sec, 52° C. for 30 sec, 72° C. for 45 sec (4 cycles)

94° C. for 15 sec, 52° C. for 30 sec (with 1° C. decrement every cycle),

72° C. for 45 sec (10 cycles)

94° C. for 15 sec, 42° C. for 30 sec, 72° C. for 45 sec (25 cycles)

72° C. for 6 min (1 cycle).

A PCR product of about 650 bp was cloned and sequenced and the deducedamino acid sequence was found to align with the putative elongasesequence compilation. The gene-specific (sense) primer

5′-ACTCGAAGCTCTTCACATGG-3 (SEQ ID NO: 7)

was synthesised and used in a further library PCR reaction with auniversal M13 forward primer

(5′-GTAAAACGACGGCCAGT-3′) (SEQ ID NO:8)

using the following conditions:

94° C. for 3 min (1 cycle)

94° C. for 15 sec, 55° C. for 30 sec, 72° C. for [lacuna] (10 cycles)

94° C. for 15 sec, 55° C. for 30 sec, 72° C. for 1 min 33 sec (with 3sec increment every cycle) (20 cycles)

72° C. for 6 min (1 cycle).

A PCR product of about 850 bp was cloned and sequenced. The two PCRproduct sequences overlapped, confirming that they were ultimatelyderived from a single gene.

Those cDNA clones isolated from cDNA libraries as described in Example 6were used for DNA sequencing according to standard methods, inparticular by the chain termination method using the ABI PRISM Big DyeTerminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer,Weiterstadt, Germany). Sequencing was carried out subsequent to plasmidrecovery from cDNA libraries via in vivo excision and retransformationof DH10_(B) on agar plates (material and protocol details fromStratagene, Amsterdam, Netherlands).

Plasmid DNA was prepared from overnight grown E. coli cultures grown inLuria-Broth medium containing ampicillin [see Sambrook et al. (1989)(Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6)].

Sequencing primers with the following nucleotide sequences were used:

5′-CAGGAAACAGCTATGACC-3′ (SEQ ID NO:9) 5′-CTAAAGGGAACAAAAGCTG-3′ (SEQ IDNO:11) 5′-TGTAAAACGACGGCCAGT-3′ (SEQ ID NO:11)

The complete nucleotide sequence of the cDNA consisted of about 1064 bp.It contained an open reading frame of 789 bp encoding 263 amino acids.The protein sequence shares just low identity or similarity with knowngenes such elongases which are required for medium-chain-length fattyacid elongation in yeast (Toke & Martin, 1996, Isolation andcharacterization of a gene affecting fatty acid elongation inSaccharomyces cerevisiae. Journal of Biological Chemistry 271,18413-18422.).

TABLE 1 Alignment of Isochrysis galbana elongase with homologoussequences. Gene M. alpina Human Mouse Yeast C. elegans Ig_ASE1 2725.5⁽¹⁾ 24.3 21⁽³⁾ 19 20.2⁽²⁾ 23.2⁽⁴⁾ 23.6⁽⁵⁾ The values in the tableare percentage identities from pairwise alignment using DNAMAN (LynnonBiosoft). Parameters used: Matrix: BLOSUM, Alignment method: optimalK-tuple: 2, Gap open: 10, Gap penalty: 4, Gap extension: 0.1; ⁽¹⁾=ELOVL4; ⁽²⁾= Helo1; ⁽³⁾= Elo1; ⁽⁴⁾= Elo2; ⁽⁵⁾= Elo3.

The sequences have been taken from human ELOVL (article 1, sequence 1),human Helo1 (article 1, sequence 2), M. alpina (Glelo, article 3), C.elegans (article 4), mouse Elov14 (article 1), yeast (sequence 3, 4, 5from articles 5 and 6).

-   1. Zhang et al., Nature Gen. 27: 89-93 (2001)-   2. Leonard et al., Biochem. J. 350: 765-770 (2000).-   3. Parker-Barnes et al., Proc. Natl. Sci. USA 97: 8284-8289, (2000).-   4. Beaudoin et al., Proc Natl. Sci. USA 97: 6421-6426. (2000)-   5. Toke and Martin, J. Biol. Chem. 271: 18413-18422 (1996)-   6. Oh et al., J. Biol. Chem. 272: 17373-17384 (1997).

Pairwise alignments of the Ig_ASE1 gene and Mortierella and mousehomologs are shown in FIG. 1 and FIG. 2.

The following parameters were used for the alignments:

Pairwise alignments: Fixed penalty: 10 Ktuple: 1 Floating penalty: 10Number of diagonals: 3 Window size: 5 Weight matrix (protein): PAM 250Gap penalty: 5

Example 6 Identification of Genes by Hybridization

Gene sequences can be used to identify homologous or heterologous genesfrom cDNA or genomic libraries.

Homologous genes (e.g. full length cDNA clones homologous to [lacuna]and homologs) can be isolated via nucleic acid hybridization using forexample cDNA libraries: Depending on the abundance of the gene ofinterest 100 000 up to 1 000 000 recombinant bacteriophages are platedand transferred to a nylon membrane. After denaturation with alkali, DNAis immobilized on the membrane by e.g. UV crosslinking. Hybridization iscarried out at high stringency conditions. In aqueous solutionhybridization and washing are performed at an ionic strength of 1 M NaCland a temperature of 68° C. Hybridization probes are generated by e.g.radioactive (³²P) nick transcription labeling (High Prime, Roche,Mannheim, Germany). Signals are detected by autoradiography.

Partially homologous or heterologous genes that are related but notidentical can be identified analogously to the above described procedureusing low stringency hybridization and washing conditions. For aqueoushybridization the ionic strength is normally kept at 1 M NaCl while thetemperature is progressively lowered from 68 to 42° C.

Isolation of gene sequences with homologies only in a distinct domain of(for example) 10-20 amino acids can be carried out by using syntheticradiolabeled oligonucleotide probes. Radio-labeled oligonucleotides areprepared by phosphorylation of the 5′-end of two complementaryoligonucleotides with T4 polynucleotide kinase. The complementaryoligonucleotides are annealed and ligated to form concatemers. Thedouble stranded concatemers are than radiolabled by for example nicktranscription. Hybridization is normally performed at low stringencyconditions using high oligonucleotide concentrations.

Oligonucleotide hybridization solution:

-   6× SSC-   0.01 M sodium phosphate-   1 mM EDTA (pH 8)-   0.5% SDS-   100 μg/ml denaturated salmon sperm DNA-   0.1% nonfat dried milk

During hybridization temperature is lowered stepwise to 5-10° C. belowthe estimated oligonucleotide Tm or down to room temperature followed bywashing steps and autoradiography. Washing is performed with extremelylow stringency such as 3 washing steps using 4× SSC. Further details aredescribed by Sambrook, J. et al. (1989), “Molecular Cloning: ALaboratory Manual”, Cold Spring Harbor Laboratory Press or Ausubel, F.M. et al. (1994) “Current Protocols in Molecular Biology”, John Wiley &Sons.

Example 7 Plasmids for Plant Transformation

For plant transformation binary vectors such as pGPTV (Becker et al.1992, Plant Mol. Biol. 20:1195-1197) or pBinAR can be used (Höfgen andWillmitzer, Plant Science 66 (1990), 221-230). Construction of thebinary vectors can be performed by ligation of the cDNA in sense orantisense orientation into the T-DNA. 5′ to the cDNA a plant promotoractivates transcription of the cDNA. A polyadenylation sequence islocated 3′ to the cDNA.

Tissue specific expression can be achieved by using a tissue specificpromotor. For example seed specific expression can be achieved bycloning the DC3 or LeB4 or USP promotor 5′ to the cDNA. Also any otherseed specific promotor element can be used. For constitutive expressionwithin the whole plant the CaMV 35S promotor can be used.

The expressed protein can be targeted to a cellular compartment using asignal peptide, for example for plastids, mitochondria or endoplasmaticreticulum (Kermode, Crit. Rev. Plant Sci. 15, 4 (1996), 285-423). Thesignal peptide is cloned 5′ in frame to the cDNA to achieve subcellularlocalization of the fusion protein.

Example 8 Transformation of Agrobacterium

Agrobacterium mediated plant transformation can be performed using forexample Agrobacterium strain C58C1 pGV2260 (Deblaere et al. 1984, Nucl.Acids Res. 13, 4777-4788) or GV3101(pMP90) (Koncz and Schell, Mol. Gen.Genet. 204 (1986), 383-396) or LBA 4404 (Clontech). Transformation canbe performed by standard transformation techniques (Deblaere et al.,Nucl. Acids. Res. 13 (1984), 4777-4788).

Example 9 Plant Transformation

Agrobacterium mediated plant transformation can be performed usingstandard transformation and regeneration techniques (Gelvin, Stanton B.;Schilperoort, Robert A, Plant Molecular Biology Manual, 2nd Ed.—Dordrecht: Kluwer Academic Publ., 1995. ISBN 0-7923-2731-4; Glick,Bernard R.; Thompson, John E., Methods in Plant Molecular Biology andBiotechnology, Boca Raton: CRC Press, 1993. —360 pp., ISBN0-8493-5164-2).

For example rapeseed can be transformed via cotyledon or hypocotyltransformation (Moloney et al., Plant cell Report 8 (1989), 238-242; DeBlock et al., Plant Physiol. 91 (1989, 694-701). Use of antibiotics foragrobacterium and plant selection depends on the binary vector and theagrobacterium strain used for transformation. Rapeseed selection isnormally performed using kanamycin as selectable plant marker.

Agrobacterium mediated gene transfer to flax can be performed using forexample a technique described by Mlynarova et al. (1994), Plant CellReport 13: 282-285.

Transformation of soybean can be performed using for example a techniquedescribed in EP 0424 047, U.S. Pat. No. 322,783 (Pioneer Hi-BredInternational) or in EP 0397 687, U.S. Pat. No. 5,376,543, U.S. Pat. No.5,169,770 (University Toledo).

Plant transformation using particle bombardment, polyethylene glycolmediated DNA uptake or via the silicon carbide fiber technique is forexample described by Freeling and Walbot in: “The maize handbook” (1993)ISBN 3-540-97826-7, Springer Verlag New York.

Example 10 In Vivo Mutagenesis

In vivo mutagenesis of microorganisms can be performed by passage ofplasmid (or other vector) DNA through E. coli or other microorganisms(e.g. Bacillus spp. or yeasts such as Saccharomyces cerevisiae) whichare impaired in their capabilities to maintain the integrity of theirgenetic information. Typical mutator strains have mutations in the genesfor the DNA repair system (e.g., mutHLS, mutD, mutT, etc.; forreference, see Rupp, W. D. (1996) DNA repair mechanisms, in: Escherichiacoli and Salmonella, p. 2277-2294, ASM: Washington). Such strains arewell known to those skilled in the art. The use of such strains isillustrated, for example, in Greener, A. and Callahan, M. (1994)Strategies 7: 32-34. Transfer of mutated DNA molecules into plants ispreferably done after selection and testing in microorganisms.Transgenic plants are generated according to various examples within theExamples section of this document.

Example 11 Assessment of the Expression of a Recombinant Gene Product ina Transformed Organism

The activity of a recombinant gene product in the transformed hostorganism has been measured on the transcriptional and/or on thetranslational level.

A useful method to ascertain the level of transcription of the gene (anindicator of the amount of mRNA available for translation of the geneproduct) is to perform a Northern blot (for reference see, for example,Ausubel et al. (1988) Current Protocols in Molecular Biology, Wiley: NewYork or within the abovementioned Examples section), in which a primerdesigned to bind to the gene of interest is labeled with a detectabletag (usually radioactive or chemiluminescent), such that when the totalRNA of a culture of the organism is extracted, run on gel, transferredto a stable matrix and incubated with this probe, the binding andquantity of binding of the probe indicates the presence and also thequantity of mRNA for this gene. This information is evidence of thedegree of transcription of the transformed gene. Total cellular RNA canbe prepared from cells, tissues or organs by several methods, allwell-known in the art, such as that described in Bormann, E. R. et al.(1992) Mol. Microbiol. 6: 317-326.

To assess the presence or relative quantity of protein translated fromthis mRNA, standard techniques, such as a Western blot, may be employed(see, for example, Ausubel et al. (1988) Current Protocols in MolecularBiology, Wiley: New York). In this process, total cellular proteins areextracted, separated by gel electrophoresis, transferred to a matrixsuch as nitrocellulose, and incubated with a probe, such as an antibody,which specifically binds to the desired protein. This probe is generallytagged with a chemiluminescent or calorimetric label which may bereadily detected. The presence and quantity of label observed indicatesthe presence and quantity of the desired mutant protein present in thecell.

Example 12 Analysis of Impact of Recombinant Proteins on the Productionof the Desired Product

The effect of the genetic modification in plants, fungi, algae, ciliatesor [lacuna] on production of a desired compound (such as fatty acids)can be assessed by growing the modified microorganisms or plants undersuitable conditions (such as those described above) and analyzing themedium and/or the cellular component for increased production of thedesired product (i.e., lipids or a fatty acid). Such analysis techniquesare well known to one skilled in the art, and include spectroscopy, thinlayer chromatography, staining methods of various kinds, enzymatic andmicrobiological methods, and analytical chromatography such as highperformance liquid chromatography (see, for example, Ullmann,Encyclopedia of Industrial Chemistry, vol. A2, p. 89-90 and p. 443-613,VCH: Weinheim (1985); Fallon, A. et al., (1987) Applications of HPLC inBiochemistry in: Laboratory Techniques in Biochemistry and MolecularBiology, vol. 17; Rehm et al. (1993) Biotechnology, vol. 3, Chapter III:Product recovery and purification, pages 469-714, VCH: Weinheim; Belter,P. A. et al. (1988) Bioseparations: downstream processing forbiotechnology, John Wiley and Sons; Kennedy, J. F. and Cabral, J. M. S.(1992) Recovery processes for biological materials, John Wiley and Sons;Shaeiwitz, J. A. and Henry, J. D. (1988) Biochemical separations, in:Ullmann's Encyclopedia of Industrial Chemistry, vol. B3, Chapter 11,pages 1-27, VCH: Weinheim; and Dechow, F. J. (1989) Separation andpurification techniques in biotechnology, Noyes Publications.).

Besides the abovementioned methods, plant lipids are extracted fromplant material as described by Cahoon et al. (1999) PNAS 96 (22):12935-12940 and Browse et al. (1986) Analytic Biochemistry 152: 141-145.Qualitative and quantitative lipid or fatty acid analysis is describedby Christie, William W., Advances in Lipid Methodology, Ayr/Scotland:Oily Press. —(Oily Press Lipid Library; 2); Christie, William W., GasChromatography and Lipids. A Practical Guide—Ayr, Scotland: Oily Press,1989 Repr. 1992. —IX, 307 pages—(Oily Press Lipid Library; 1); “Progressin Lipid Research, Oxford: Pergamon Press, 1(1952)—16(1977) under thetitle: Progress in the Chemistry of Fats and Other Lipids.

In addition to the measurement of the final product of fermentation, itis also possible to analyze other components of the metabolic pathwaysutilized for the production of the desired compound, such asintermediates and by-products, to determine the overall efficiency ofproduction of the compound. Analysis methods include measurements ofnutrient levels in the medium (e.g., sugars, hydrocarbons, nitrogensources, phosphates, and other ions), measurements of biomasscomposition and growth, analysis of the production of common metabolitesof biosynthetic pathways, and measurement of gases produced duringfermentation. Standard methods for these measurements are outlined inApplied Microbial Physiology, A Practical Approach, P. M. Rhodes and P.F. Stanbury, eds., IRL Press, p. 103-129; 131-163; and 165-192 (ISBN:0199635773) and references cited therein.

One example is the analysis of fatty acids (abbreviations: FAME, fattyacid methyl ester; GC-MS, gas-liquid chromatography-mass spectrometry;TAG, triacylglycerol; TLC, thin-layer chromatography).

Unequivocal proof for the presence of fatty acid products can beobtained by the analysis of recombinant organisms following standardanalytical procedures: GC, GC-MS or TLC as variously described byChristie and references therein (1997, in: Advances on LipidMethodology—Fourth ed.: Christie, Oily Press, Dundee, 119-169; 1998,gas-chromatography-mass spectrometry methods, Lipids 33:343-353).

Material to be analyzed can be disintegrated via sonification, glassmilling, liquid nitrogen and grinding or via other applicable methods.The material has to be centrifuged after disintegration. The sediment isresuspended in Aqua dest, heated for 10 min at 100° C., cooled on iceand centrifuged followed by extraction in 0.5 M sulfuric acid inmethanol containing 2% dimethoxypropane for 1 h at 90° C., leading tohydrolyzed oil and lipid compounds resulting in transmethylated lipids.These fatty acid methyl esters are extracted in petroleum ether andfinally subjected to GC analysis using a capillary column (Chrompack,WCOT Fused Silica, CP-Wax-52 CB, 25 m, 0.32 mm) at a temperaturegradient between 170° C. and 240° C. for 20 min and 5 min at 240° C. Theidentity of the resulting fatty acid methyl esters has to be defined bythe use of standards available form commercial sources (i.e. Sigma).

In the case of fatty acids where standards are not available moleculeidentity has to be shown via derivatization and subsequent GC analysis.For example the localization of triple bond fatty acids has to be shownvia GC-MS after derivatization with 4,4-dimethoxyoxazoline derivatives(Christie, 1998, see above).

Example 13 Expression Products in Heterologous Microbial Systems

Strains, Growth Conditions and Plasmids

Escherichia coli strain XL1 Blue MRF′ kan (Stratagene) was used forsubcloning the new elongase Ig_ASE1 from Isochrysis galbana. Forfunctional expression of this gene we used the Saccharomyces cerevisiaestrain INVSc 1 (Invitrogen Co.). E. coli was grown in Luria-Bertanibroth (LB, Duchefa, Haarlem, The Netherlands) at 37° C. When neccessary,ampicillin (100 mg/liter) was added and 1.5% (w/v) agar (Difco) wasincluded for solid LB media. S. cerevisiae was grown at 30° C. either inYPG-medium or in complete minimal dropout uracil medium (CMdum; see in:Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J.G., Smith, J. A., Struhl, K., Albright, L. B., Coen, D. M., and Varki,A. (1995), Current Protocols in Molecular Biology, John Wiley & Sons,New York.) containing either 2% (w/v) raffinose or glucose. For solidmedia 2% (w/v) Bacto™ agar (Difco) was included. Plasmids used forcloning and expression were pUC 18 (Pharmacia) and pYES2 (InvitrogenCo.).

Example 14 Cloning and Expression of an ALA-PUFA Specific Elongase (ASEGene) from Isochrysis Galbana in Yeast

a) Cloning Procedures

For expression in yeast, the Isochrysis galbana gene Ig_ASE1 was firstmodified to create restriction sites and the yeast consensus sequencefor highly efficient translation (Kozak, M. (1986). Point mutationsdefine a sequence flanking the AUG initiator codon that modulatestranslation by eukaryotic ribosomes (Cell 44, 283-292.). A site adjacentto the start codon was introduced. For amplification of the open readingframe a pair of primers complementary to its 5′- and 3′-end weresynthesized.

(SEQ ID NO:12) Forward primer: 5′-GGTACCATGGCCCTCGCAAACGA-3′ (SEQ IDNO:13) Reverse primer: 5′-TAGGACATCCACAATCCAT-3′

The PCR reaction was performed with plasmid-DNA as template in aThermocycler (Biometra) using Pfu DNA polymerase (Stratagene) and thefollowing temperature program: 3 min. at 96° C. followed by 25 cycleswith 30 s at 96° C., 30 s at 55° C. and 3 min. at 72° C., 1 cycle with10 min. at 72° C. and stop at 4° C.

The correct size of the amplified DNA fragment of about 800 bp wasconfirmed by Agarose-TBE gel electrophoresis. The amplified DNA wasextracted from the gel with the QIAquick Gel Extraktion Kit (QIAGEN) andligated into the T/A-site of the vector pCR 21 (Invitrogen) using theSure Clone Ligation Kit (Pharmacia). After transformation of E. coli XL1Blue MRF′ kan a DNA mini-preparation (Riggs, M. G. & McLachlan, A.(1986), A simplified screening procedure for large numbers of plasmidmini-preparation. BioTechniques 4, 310-313.) was performed with 24ampicillin-resistant transformants and positive clones were identifiedby BamHI restriction analysis. The sequence of the cloned PCR productwas confirmed by resequencing using the ABI PRISM Big Dye TerminatorCycle Sequencing Ready Reaction Kit (Perkin-Elmer, Weiterstadt).

The plasmid-DNA of pCR-ASE1 was further restricted with KpnI/SacI andthe resulting DNA fragment was ligated into the same restriction site ofthe dephosphorylated yeast-E. coli shuttle-vector pYES2 resulting inpY2ASE1. After transformation of E. coli and DNA mini-preparation fromthe transformants, the orientation of the DNA fragment within the vectorwas checked. One clone was grown for DNA maxi-preparation with theNucleobond® AX 500 Plasmid-DNA Extraction Kit (Macherey-Nagel,Düringen).

Saccharomyces cerevisiae INVSc1 was transformed with pY2ASE1 and pYES2by a modified PEG/lithium acetate protocol (Ausubel et al., 1995). Afterselection on CMdum agar plates containing 2% glucose, four pY2ASE1transformants and one pYES2 transformant were chosen for furthercultivation and functional expression.

b) Functional Expression of Elongase Activity in Yeast

Preculture:

20 ml of CMdum liquid medium containing 2% (w/v) raffinose wereinoculated with the transgenic yeast clones (pY2ASE1a-d, pYES2) andgrown for 3 days at 30° C., 200 rpm until an optical density at 600 nm(OD₆₀₀) of 1.5-2 was reached.

Main Culture:

For expression 20 ml Cmdum liquid medium with 2% raffinose and 1% (v/v)Tergitol NP-40 was supplemented with the fatty acid to be tested to afinal concentration of 0.003% (w/v). The media were inoculated with theprecultures to an OD₆₀₀ of 0.05. The expression was induced at an OD₆₀₀of 0.2 with 2% (w/v) galactose for 16 h, after which time the cultureshad reached an OD₆₀₀ of 0.8-1.2.

c) Fatty Acid Analysis

The total fatty acids were extracted from yeast cultures and analyzed bygas chromatography. For this, cells from 5 ml culture were harvested bycentrifugation (1000×g, 10 min., 4° C.) and washed once with 100 mMNaHCO₃, pH 8.0 to remove residual medium and fatty acids. Forpreparation of the fatty acid methyl esters (FAMES) the cell pelletswere treated with 1 N methanolic H₂SO₄ and 2% (v/v) dimethoxypropane for1 h at 80° C. The FAMES were extracted twice with 2 ml petroleum ether,washed once with 100 mM NaHCO₃, pH 8.0 and once with distilled water anddried with Na₂SO₄. The organic solvent was evaporated under a stream ofargon and the FAMES were dissolved in 50 μl of petroleum ether. Thesamples were separated on a ZEBRON ZB-Wax capillary column (30 m, 0.32mm, 0.25 μm; Phenomenex) in a Hewlett Packard 6850 gas chromatographwith a flame ionization detector. The oven temperature was programmedfrom 70° C. (1 min. hold) to 200° C. at a rate of 20° C./min., then to250° C. (5 min. hold) at a rate of 5° C./min and finally to 260° C. at arate of 5° C./min. Nitrogen was used as carrier gas (4.5 ml/min. at 70°C.). The fatty acids were identified by comparison with retention timesof FAME standards (SIGMA).

Fatty acid patterns of transgenic yeast strains are shown in Tab. 2

TABLE 2 Fatty acid patterns in mol % of transgenic yeast strains +LA+ALA +GLA −Substrate (18:2 n − 6) (18:3 n − 3) (18:3 n − 6) Induction+gal −gal +gal −gal +gal −gal +gal −gal 16:0 28.7 30.2 27.0 28.9 26.628.9 30.0 31.0 16:1 n − 9 41.6 42.4 30.7 25.4 30.1 26.4 24.3 24.6 18:06.8 6.1 5.7 5.8 6.3 6.3 6.8 6.2 18:1 n − 9 22.9 21.3 16.5 13.4 18.4 16.614.7 13.4 18:2 n − 6* — — 11.0 26.5 — — — — 18:3 n − 6* — — — — — — 24.224.8 18:3 n − 3* — — — — 10.2 21.8 — — 20:2 n − 6 — — 9.1 — — — — — 20:3n − 3 — — — — 8.4 — — — % Elongation 0 — 45.3 — 45.2 — 0 —Explanation to Tab. 2:

Fatty acid elongation of different substrates supplied to transgenicyeast containing pY2ASE1. Exogenous fatty acids supplied as substratesfor elongation are indicated by an asterisk [*]. The values given areexpressed as mol % of total fatty acid methyl esters identified by GCand FID. In the case of elongated substrates, this is also expressed asa % conversion. Expression of the ASE1 transgene was induced by theaddition of galactose. Only C18 substrates with a double bond at the Δ9position were elongated by the ASE1 open reading frame. All valuesrepresent the mean of three separate experiments.

GC analysis of FAMES prepared from total lipids of the yeaststransformed with pY2ASE1 and grown in the presence of differentexogenous fatty acids (ALA, GLA, LA), and their fatty acid patterns areshown in mol % in Table 1. The incorporation of GLA does not yield anyelongation product di-homo-GLA (20:3 d8,11,14) while ALA is elongated toyield C20:3 d11,14,17, and LA is elongated to yield C20:2 d11, 14.

The transgenic yeast clones transformed with pY2ASE1 and supplied withexogenous substrates show an additional peak in the gas chromatogram(identified by an asterisk [*] in FIGS. 3A-D), which has been identifiedby comparison of retention times as the fed/incorporated fatty acid. Agas chromatography/mass spectroscopy can give additional support toconfirm its identity.

FIG. 3A-D shows essentially a GC graph of data presented in Table 2.Explanation to FIG. 3A-D:

GC chromatograms of fatty acid methyl esters extracted from transgenicyeast containing pY2ASE1. Yeast cultures were grown in the presence(indicated by an asterisk) or absence of exogenous fatty acids.Exogenous fatty acids (in the form of sodium salts) were LA (linoleicacid; 18:2 Δ^(9, 12); 18:2 n-6, see FIG. 3B), ALA (α-linolenic acid;18:3 Δ^(9, 12, 15); 18:3 n-3, see FIG. 3A), GLA (γ-linolenic acid; 18:3Δ^(6, 9, 12); 18:3 n-6, see FIG. 3C) or no substrate (FIG. 3D). FIG. 3Brepresents expression of the ASE1 ORF as induced by the addition ofgalactose. After 24 h, yeast cells were harvested by centrifugation,washed to remove exogenous substrate and methylated. Fatty acid methylesters were separated and detected using standard methods and peaksidentified by comigration of known standards. It is clear that Ig_ASE1encodes a Δ9-C₁₈-PUFA-specific elongating activity. Identified productsshow that nucleotide sequence of Ig_ASE1 codes for a Δ9-selective C₁₈fatty acid elongase from the alga Isochrysis galbana, which leads to theformation of new fatty acids in transgenic yeasts.

Further feeding experiments with several other fatty acids can beperformed to confirm the substrate selectivity of this elongase infurther detail.

Example 15 Purification of the Desired Product from TransformedOrganisms in General

Recovery of the desired product from plant material or fungi, algae,ciliate cells or supernatant of the above-described cultures can beperformed by various methods well known in the art. If the desiredproduct is not secreted from the cells, the cells can be harvested fromthe culture by low-speed centrifugation, the cells can be lysed bystandard techniques, such as mechanical force or sonification. Organs ofplants can be separated mechanically from other tissue or organs.Following homogenization cellular debris is removed by centrifugation,and the supernatant fraction containing the soluble proteins is retainedfor further purification of the desired compound. If the product issecreted from desired cells, then the cells are removed from the cultureby low-speed centrifugation, and the supernatant fraction is retainedfor further purification.

The supernatant fraction from each purification method is subjected tochromatography with a suitable resin, in which the desired molecule iseither retained on the chromatography resin while many of the impuritiesin the sample are not, or where the impurities are retained by the resinwhile the sample is not. Such chromatography steps may be repeated asnecessary, using the same or different chromatography resins. Oneskilled in the art would be well-versed in the selection of appropriatechromatography resins and in their most efficacious application for aparticular molecule to be purified. The purified product may beconcentrated by filtration or ultrafiltration, and stored at atemperature at which the stability of the product is maximized.

There are a wide array of purification methods known in the art and thepreceding method of purification is not meant to be limiting. Suchpurification techniques are described, for example, in Bailey, J. E. &Ollis, D. F. Biochemical Engineering Fundamentals, McGraw-Hill: N.Y.(1986).

The identity and purity of the isolated compounds may be assessed bytechniques standard in the art. These include high-performance liquidchromatography (HPLC), spectroscopic methods, staining methods, thinlayer chromatography, NIRS, enzymatic assays, or microbiological assays.Such analysis methods are reviewed in: Patek et al. (1994) Appl.Environ. Microbiol. 60: 133-140; Malakhova et al. (1996) Biotekhnologiya11: 27-32; and Schmidt et al. (1998) Bioprocess Engineer. 19: 67-70.Ullmann's Encyclopedia of Industrial Chemistry, (1996) vol. A27, VCH:Weinheim, p. 89-90, p. 521-540, p. 540-547, p. 559-566, 575-581 and p.581-587; Michal, G. (1999) Biochemical Pathways: An Atlas ofBiochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. etal. (1987) Applications of HPLC in Biochemistry in: LaboratoryTechniques in Biochemistry and Molecular Biology, vol. 17.

EQUIVALENTS

Those skilled in the art will recognize, or will be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the claims.

1. An isolated nucleic acid comprising a nucleotide sequence whichencodes a polypeptide which elongates α-linolenic acid(C_(18:3 d9, 12, 15)) by at least two carbon atoms whereas γ-linolenicacid (C_(18:3 d6, 9, 12)) is not elongated, selected from the groupconsisting of: a) the nucleic acid sequence of SEQ ID NO: 1; b) anucleic acid sequence which encodes the polypeptide of SEQ ID NO: 2; andc) a derivative of the nucleic acid sequence of SEQ ID NO: 1, whichencodes a polypeptide having at least 95% homology with the nucleic acidsequence encoding the amino acid sequence of SEQ ID NO:
 2. 2. Theisolated nucleic acid as claimed in claim 1, which is derived from aplant.
 3. The isolated nucleic acid as claimed in claim 1, which isderived from the genus Isochrysis.
 4. A gene construct comprising theisolated nucleic acid sequence as claimed in claim 1, functionallylinked to one or more regulatory signals.
 5. The gene construct asclaimed in claim 4, further comprising a fatty acid biosynthesis gene.6. The gene construct as claimed in claim 5, wherein the fatty acidbiosynthesis gene is selected from the group consisting of Δ19-, Δ17-,Δ15-, Δ12-, Δ9-, Δ8-, Δ6-, Δ5-, Δ4-desaturase, hydroxylase, elongase,Δ12-acetylenase, Acyl-ACP-thioesterasen, β-ketoacyl-ACP-synthase andβ-ketoacyl-ACP-reductase.
 7. A vector comprising a nucleic acid asclaimed in claim 1, or a gene construct comprising said nucleic acid. 8.A plant cell comprising at least one heterologous nucleic acid asclaimed in claim
 1. 9. A plant comprising the plant cell of claim
 8. 10.The plant of claim 9, which is a transgenic plant.
 11. An antisensenucleotide sequence which is fully complementary to a nucleotidesequence selected from the group consisting of: a) the nucleic acidsequence of SEQ ID NO: 1; b) a nucleic acid sequence which encodes thepolypeptide of SEQ ID NO: 2; and c) a derivative of the nucleic acidsequence of SEQ ID NO: 1, which encodes a polypeptide having at least95% homology with the nucleic acid sequence encoding the amino acidsequence of SEQ ID NO: 2; and wherein said polypeptide elongatesα-linolenic acid (C_(18:3 d9, 12, 15)) by at least two carbon atomswhereas γ-linolenic acid (C_(18:3 d6, 9, 12)) is not elongated.
 12. Anisolated nucleic acid comprising SEQ ID NO:
 1. 13. An amino acidsequence comprising SEQ ID NO: 2.